U.S. patent application number 17/052960 was filed with the patent office on 2021-05-06 for antibodies specific to human nectin4.
The applicant listed for this patent is NECTIN THERAPEUTICS LTD., YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD.. Invention is credited to Stipan JONJIC, Ofer MANDELBOIM, Adi RECHES, Pinchas TSUKERMAN.
Application Number | 20210130459 17/052960 |
Document ID | / |
Family ID | 1000005343964 |
Filed Date | 2021-05-06 |
United States Patent
Application |
20210130459 |
Kind Code |
A1 |
MANDELBOIM; Ofer ; et
al. |
May 6, 2021 |
ANTIBODIES SPECIFIC TO HUMAN NECTIN4
Abstract
The present invention provides monoclonal antibodies that
recognize human Nectin4 with high affinity and specificity and
inhibit its binding to T cell immunoreceptor with Ig and ITIM
domains (TIGIT). The present invention further provides
pharmaceutical compositions comprising the antibodies and methods
for their use in cancer immunotherapy and in diagnosis.
Inventors: |
MANDELBOIM; Ofer; (Shoham,
IL) ; RECHES; Adi; (Koranit, IL) ; JONJIC;
Stipan; (Rijeka, HR) ; TSUKERMAN; Pinchas;
(Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM LTD.
NECTIN THERAPEUTICS LTD. |
Jerusalem
Jerusalem |
|
IL
IL |
|
|
Family ID: |
1000005343964 |
Appl. No.: |
17/052960 |
Filed: |
May 6, 2019 |
PCT Filed: |
May 6, 2019 |
PCT NO: |
PCT/IL2019/050508 |
371 Date: |
November 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62668824 |
May 9, 2018 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2803 20130101; A61K 45/06 20130101; A61K 2039/505
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61P 35/00 20060101
A61P035/00 |
Claims
1-42. (canceled)
43. An isolated monoclonal antibody which binds to Nectin4, or an
antibody fragment thereof comprising at least the antigen binding
portion, wherein the isolated antibody or antibody fragment is
capable of inhibiting the binding of Nectin4 to T cell
immunoreceptor with Ig and ITIM domains (TIGIT), wherein the
isolated antibody or antibody fragment comprises: (i) three
complementarity determining regions (CDRs) of a heavy chain (HC)
variable region comprising SEQ ID NO: 39 and three CDRs of a light
chain (LC) variable region comprising SEQ ID NO: 40, or an analog
or derivative thereof having at least 90% sequence identity with
said antibody or fragment sequence; (ii) three CDRs of a heavy
chain (HC) variable region comprising SEQ ID NO: 35 and three CDRs
of a light chain (LC) variable region comprising SEQ ID NO: 36, or
an analog or derivative thereof having at least 90% sequence
identity with said antibody or fragment sequence; or (iii) three
CDRs of a heavy chain variable region comprising SEQ ID NO: 37 and
three CDRs of a light chain variable region comprising SEQ ID NO:
38, or an analog or derivative thereof having at least 90% sequence
identity with said antibody or fragment sequence.
44. The isolated monoclonal antibody or the antibody fragment
according to claim 43, comprising a CDR set selected from the group
consisting of: (i) a set of six CDRs wherein: HC CDR1 is SYYIH (SEQ
ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3
is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO:
28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT
(SEQ ID NO: 30); (ii) a set of six CDRs wherein: HC CDR1 is AYNIH
(SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC
CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSSVSYMH (SEQ ID
NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is
FQGSGSPYT (SEQ ID NO: 14); and (iii) a set of six CDRs wherein: HC
CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID
NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is
KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19),
and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).
45. The isolated monoclonal antibody or the antibody fragment
according to claim 43, comprising a heavy chain and a light chain,
wherein the heavy chain comprises SEQ ID NO: 39 and the light chain
comprises SEQ ID NO: 40.
46. The isolated monoclonal antibody or the antibody fragment
according to claim 43, comprising a heavy chain and a light chain,
wherein the heavy chain comprises SEQ ID NO: 35 and the light chain
comprises SEQ ID NO: 36.
47. The isolated monoclonal antibody or the antibody fragment
according to claim 43, comprising a heavy chain and a light chain,
wherein the heavy chain comprises SEQ ID NO: 37 and the light chain
comprises SEQ ID NO: 38.
48. An isolated monoclonal fragment according to claim 43, wherein
the fragment is a single chain Fv (scFv).
49. The isolated monoclonal fragment of claim 48, comprising a
sequence selected from the group consisting of SEQ ID NO: 32 and
SEQ ID NO: 34, or a variant thereof having at least 90% sequence
similarity with said sequences.
50. The isolated monoclonal antibody or antibody fragment according
to claim 43, wherein the monoclonal antibody binds to human Nectin4
with an affinity of 10.sup.-9M to 10.sup.-10M.
51. A polynucleotide sequence encoding at least one region of a HC
or a LC sequence of a monoclonal antibody or antibody fragment
according to claim 43.
52. The polynucleotide sequence of claim 51, encoding: (a) a
monoclonal antibody heavy chain variable region, wherein the
polynucleotide sequence comprises a sequence selected from the
group consisting of SEQ ID NO: 45, SEQ ID NO: 41, SEQ ID NO: 43,
and a variant thereof having at least 90% identity to said
sequences; or (b) a monoclonal antibody light chain variable
region, wherein the polynucleotide sequence is selected from the
group consisting of: SEQ ID NO: 46, SEQ ID NO: 42, SEQ ID NO: 44,
and a variant thereof having at least 90% identity to said
sequences.
53. A plasmid or cell comprising at least one polynucleotide
sequence according to claim 51.
54. A pharmaceutical composition comprising as an active
ingredient, at least one isolated antibody or fragment thereof,
according to claim 43 and a pharmaceutical acceptable excipient,
diluent, salt, or carrier.
55. The pharmaceutical composition of claim 54 wherein the
monoclonal antibody is not conjugated to a cytotoxic moiety.
56. A method of treating a viral infection in a subject, comprising
administering to a subject in need thereof, a pharmaceutical
composition according to claim 54.
57. A method of treating cancer, comprising administering to a
subject in need thereof, a pharmaceutical composition according to
claim 54.
58. The method of claim 57, further comprising an additional
anti-cancer therapy selected from surgery, chemotherapy,
radiotherapy, and immunotherapy.
59. The method of 57, further comprising administering to said
subject an additional immuno-modulator, activated lymphocyte cell,
kinase inhibitor, chemotherapeutic agent or any other anti-cancer
agent.
60. The method of claim 57, wherein the antibody or fragment
thereof inhibits the interaction between Nectin4 and Nectin1 on
tumor cells.
61. A method of diagnosing a cancer in a subject, the method
comprising contacting a biological sample with an antibody or
antibody fragment according to claim 43.
62. A chimeric antigen receptor (CAR) comprising at least one
antibody or antibody fragment according to claim 43.
63. The CAR according to claim 62, comprising a sequence selected
from the group consisting of SEQ ID NOs: 32 and 34, a transmembrane
domain, and an intracellular T or NK cell signaling domain.
64. A population of T-cells or NK-cells engineered to express the
CAR according to claim 62.
65. A method of treating cancer comprising administering to a
subject in need thereof, at least one cell expressing the CAR
according to claim 62.
Description
FIELD OF THE INVENTION
[0001] The invention is in the field of immunotherapy and relates
to antibodies and fragments thereof which bind to the human protein
Nectin4, to polynucleotide sequences encoding these antibodies and
to cells producing these antibodies. The invention further relates
to therapeutic and diagnostic compositions comprising these
antibodies and to methods of treating and diagnosing diseases,
particularly cancer, using these antibodies.
BACKGROUND OF THE INVENTION
[0002] Immunotherapy is one of the most promising advancements made
in the past decade in cancer treatment. Cancer immunotherapy is
utilized for generating and augmenting an anti-tumor immune
response, e.g., by treatment with antibodies specific to antigens
on tumor cells, with fusions of antigen presenting cells with tumor
cells, or by activation of anti-tumor T cells. The ability of
recruiting immune cells (e.g. T cells) against tumor cells in a
patient provides a therapeutic modality of fighting cancer types
and metastasis that so far were considered incurable.
[0003] T cell mediated immune response includes multiple sequential
steps regulated by a balance between co-stimulatory and
co-inhibitory signals that control the magnitude of the immune
response. The inhibitory signals, referred to as immune
checkpoints, are crucial for the maintenance of self-tolerance and
for limiting immune-mediated collateral tissue damage. These
signals change as an infection or immune provocation is cleared,
worsens, or persists, and these changes affect and re-shape the
immune response.
[0004] The expression of immune checkpoint proteins can be
regulated by tumors. For example, upregulation of programmed death
ligand-1 (PD-L1) on the cancer cell surface allows them to evade
the host immune system by inhibiting T cells via binding to PD-1
that might otherwise attack these tumor cells. Thus, immune
checkpoints represent significant barriers to activation of
functional cellular immunity against cancer. Accordingly,
antagonistic antibodies specific for inhibitory ligands on immune
cells are considered viable anti-cancer agents and they are being
used in cancer treatment (e.g. Nivolumab and Pembrolizumab).
Another example for immune checkpoint molecule is "T cell
immunoreceptor with Ig and ITIM domains" (TIGIT). TIGIT is a
co-inhibitory molecule expressed on various immune cells including
T cells and Natural Killer cells (NK cells). TIGIT binds with high
affinity to polio-virus receptor (PVR, CD155). Monoclonal
antibodies (mAbs) specific for TIGIT are disclosed for example in
WO 2016/028656 and WO 2017/037707.
[0005] Poliovirus receptor (PVR), is a transmembrane glycoprotein
involved in mediating cell adhesion to extracellular matrix
molecules. PVR is a known tumor antigen and a target for
therapeutic interventions. Blocking of PVR on tumor cells is
anticipated to reduce viability of tumor cells. PVR has also a
critical role in angiogenesis and metastasis. Several patent
applications including U.S. Patent Application No. 20070041985,
U.S. Patent Application No. 20090215175 and WO 2017149538, disclose
molecules and antibodies that specifically bind to PVR and their
use against cancer.
[0006] Nectin Cell Adhesion Molecule 4 (Nectin4), also termed
poliovirus receptor-related 4 (PVRL4), is a type I transmembrane
protein and member of the Nectin family of related
immunoglobulin-like adhesion molecules. Nectin4 is a tumor
associated marker for many tumors including lung, breast, colon,
and ovarian cancers.
[0007] Chailta-Eid at al. 2016 (Cancer Res. 2016; 76:3003-13)
discloses anti Nectin4 (Enfortumab) antibody-drug conjugate as a
highly potent therapeutic agent in multiple preclinical cancer
models. The antibody, conjugated with the microtubule inhibitor
vedotin, binds human, as well as rat and monkey nectin4 and
inhibits growth of several cell lines and xenografts that express
nectin4.
[0008] Despite of the success made in cancer immunotherapy, there
is still an unmet need for additional approaches, and more
effective and specific agents and drug combinations to potentiate
cells of the immune system to attack tumor cells. One such approach
involves inhibiting Nectin4 binding to TIGIT by specific monoclonal
antibodies.
SUMMARY OF THE INVENTION
[0009] The present invention provides antibodies and fragments
thereof which bind to the human protein Nectin4, to polynucleotide
sequences encoding these antibodies and to cells producing these
antibodies. The present invention is based in part on the discovery
that Nectin4, previously known as a receptor for measles virus and
a tumor antigen, is a ligand for the immune-inhibitory molecule
TIGIT and therefore proposed for the first time as a target for
inhibition of the suppressive effect of TIGIT on anti-cancer
immunity. The present invention further provides in some
embodiments chimeric antigen receptors (CARs) comprising a binding
site to Nectin4.
[0010] The present invention provides highly effective monoclonal
antibodies (mAbs) specific to human Nectin4, which not only block
the interaction between Nectin4 and the inhibitory receptor TIGIT,
but also have direct effect on target cells expressing this
receptor. These antibodies, having binding constant to Nectin4 in
the sub-nanomolar range, reverse TIGIT inhibition of the immune
system and directly enhance elimination of tumor cells, without
being conjugated to any toxin or anti-tumor agent. The antibodies
of the present invention are therefore useful for inhibition of the
interaction between Nectin4 on target cells and TIGIT on immune
cells, for example in cancer immunotherapy. Moreover, some intact
antibodies described herein induce ADCC (antibody-dependent
cell-mediated cytotoxicity) activity. Nectin4 is specifically
overexpressed on tumor cells. ADCC activity induction together with
the high affinity of the antibodies of the present invention to
Nectin4 and their ADCC activity, makes them ideal candidates for
immunotherapy.
[0011] The present invention provides antibodies and fragments
thereof that recognize the protein Nectin4, prevent its binding to
the protein TIGIT and inhibit suppressive activity on lymphocytes
such as natural killer (NK) cells and T-cells. The anti-Nectin4
antibodies disclosed herein are capable of binding to Nectin4
present on target cells such as cancer cells.
[0012] The antibodies and fragment of the present invention are
characterized by having unique sets of complementarity-determining
regions (CDR) sequences, high affinity and high specificity to
human Nectin4, and are useful in cancer immunotherapy for combating
tumor immune evasion, as stand-alone therapy and in combination
with other anti-cancer agents. The antibodies are also useful in
preventing viral infections, in particular, prevention of measles
infection.
[0013] It is now disclosed that the high affinity anti-Nectin4
antibodies disclosed herein block TIGIT-Nectin4 interaction and
restore T and NK cells activity.
[0014] Some of the monoclonal antibodies of the present invention
are also capable of blocking the interaction between Nectin4 and
Nectin1, indicating their ability to interfere with invasiveness of
tumors expressing Nectin4. In addition, anti Nectin4 mAbs were able
to induce NK cell activation in most target cells. Advantageously,
the anti-Nectin4 mAbs according to the invention have direct effect
on target cancer cells, inducing their killing without the need of
NK cells and/or of a toxin. It is further disclosed that the
anti-Nectin4 antibodies of the invention had no blocking effect on
signaling of the co-stimulatory receptors such as DNAM1, therefore
they are expected to have no deleterious effects on other immune
induction signals.
[0015] Interestingly, despite high sequence similarity between
human and rodent Nectin4 sequences, some of the antibodies of the
present invention are highly specific to human Nectin4 and do not
bind rodent Nectin4.
[0016] Some of the anti-Nectin4 mAbs described herein were able to
reduce tumor cells viability in an immune independent manner by
blocking of Nectin4 on tumor cells. In some embodiments, the
Nectin4 antibodies described herein inhibit proliferation of tumor
cells, via immune independent interference with binding to Nectin1
on tumor cells, without being bound to any toxic molecule.
[0017] According to one aspect, the present invention provides an
isolated monoclonal antibody (mAb), or an antibody fragment thereof
comprising at least the antigen binding portion, which specifically
binds to human Nectin4 and inhibits its binding to TIGIT.
[0018] The present invention also provides a mAb or antibody
fragment thereof, capable of inhibiting the binding of human
Nectin4 to human TIGIT, for use in treatment of cancer, together
with T-cell lymphocytes and/or natural killer (NK) cells.
[0019] According to some embodiments, the mAb is not conjugated to
any toxin or anti-tumor agent.
[0020] According to some embodiments, the isolated antibody or
antibody fragment comprises a set of six complementarity
determining region (CDR) sequences selected from the group
consisting of: [0021] i. three CDRs of a heavy chain (HC) variable
region comprising SEQ ID NO: 22 and three CDRs of a light chain
(LC) variable comprising SEQ ID NO: 24, or an analog or derivative
thereof having at least 90% sequence identity with said antibody or
fragment sequence; [0022] ii. three CDRs of a HC variable region
comprising SEQ ID NO: 2 and three CDRs of a LC variable comprising
SEQ ID NO: 4, or an analog or derivative thereof having at least
90% sequence identity with said antibody or fragment sequence; and
[0023] iii. three CDRs of a HC variable region comprising SEQ ID
NO: 6 and three CDRs of a LC variable region comprising SEQ ID NO:
8, or an analog or derivative thereof having at least 90% sequence
identity with said antibody or fragment sequence.
[0024] According to some embodiments, the isolated antibody or
antibody fragment comprises a set of six CDR sequences selected
from the group consisting of: [0025] iv. three CDRs of a HC
variable region comprising SEQ ID NO: 39 and three CDRs of a LC
variable comprising SEQ ID NO: 40, or an analog or derivative
thereof having at least 90% sequence identity with said antibody or
fragment sequence; [0026] v. three CDRs of a HC variable region
comprising SEQ ID NO: 35 and three CDRs of a LC variable comprising
SEQ ID NO: 36, or an analog or derivative thereof having at least
90% sequence identity with said antibody or fragment sequence; and
[0027] vi. three CDRs of a HC variable region comprising SEQ ID NO:
37 and three CDRs of a LC variable region comprising SEQ ID NO: 38,
or an analog or derivative thereof having at least 90% sequence
identity with said antibody or fragment sequence.
[0028] There are several methods known in the art for determining
the CDR sequences of a given antibody molecule, but there is no
standard unequivocal method. Determination of CDR sequences from
antibody heavy and light chain variable regions can be made
according to any method known in the art, including but not limited
to the methods known as KABAT, Chothia and IMGT. A selected set of
CDRs may include sequences identified by more than one method,
namely, some CDR sequences may be determined using KABAT and some
using IMGT, for example. According to some embodiments, the CDR
sequences of the mAb variable regions are determined using the IMGT
method.
[0029] According to some embodiments, the isolated monoclonal
antibody or fragment comprises the CDR sequences of a monoclonal
antibody denoted hNec4.11 (or Nectin4.11, or clone 11), namely, the
three CDR sequences contained in heavy chain variable region set
forth in SEQ ID NO: 39 and the three CDR sequences contained in
light chain variable region set forth in SEQ ID NO: 40.
[0030] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises heavy chain CDR1
comprising the sequence SYYIH (SEQ ID NO: 25). According to some
embodiments, the isolated monoclonal antibody or the antibody
fragment comprises heavy chain CDR2 comprising the sequence
WIYPGNVNTKYNERFKG (SEQ ID NO: 26). According to some embodiments,
the isolated monoclonal antibody or the antibody fragment comprises
heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO:
27).
[0031] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) HC CDR1 comprising
the sequence SYYIH (SEQ ID NO: 25); (ii) HC CDR2 comprising the
sequence: WIYPGNVNTKYNERFKG (SEQ ID NO: 26); and (iii) HC CDR3
comprising the sequence: SNPYVMDY (SEQ ID NO: 27).
[0032] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises light chain CDR1
comprising the sequence KASQSVNNDVA (SEQ ID NO: 28). According to
some embodiments, the isolated monoclonal antibody or the antibody
fragment comprises light chain CDR2 comprising the sequence YASNRFT
(SEQ ID NO: 29). According to some embodiments, the isolated
monoclonal antibody or the antibody fragment comprises light chain
CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 30).
[0033] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) LC CDR1 comprising
the sequence KASQSVNNDVA (SEQ ID NO: 28); (ii) LC CDR2 comprising
the sequence: YASNRFT (SEQ ID NO: 29); and (iii) HC CDR3 comprising
the sequence: QQAYRSPYT (SEQ ID NO: 30).
[0034] According to some specific embodiments the isolated
monoclonal antibody or fragment comprises heavy chain CDR1 sequence
comprising the sequence: SYYIH (SEQ ID NO: 25), heavy chain CDR2
comprising the sequence: WIYPGNVNTKYNERFKG (SEQ ID NO: 26), heavy
chain CDR3 comprising the sequence: SNPYVMDY (SEQ ID NO: 27), light
chain CDR1 comprising the sequence: KASQSVNNDVA (SEQ ID NO: 28),
light chain CDR2 comprising the sequence: YASNRFT (SEQ ID NO: 29),
and light chain CDR3 comprising the sequence: QQAYRSPYT (SEQ ID NO:
30), or analogs thereof comprising no more than 5% amino acid
substitution, deletion and/or insertion in the hypervariable region
(HVR) sequence.
[0035] According to some specific embodiments, the isolated
monoclonal antibody or fragment comprises a set of six CDR
sequences consisting of: [0036] i. heavy chain CDR1 having a
sequence set forth in SEQ ID NO: 25; [0037] ii. heavy chain CDR2
having a sequence set forth in SEQ ID NO: 26; [0038] iii. heavy
chain CDR3 having a sequence set forth in SEQ ID NO: 27; [0039] iv.
light chain CDR1 having a sequence set forth in SEQ ID NO: 28;
[0040] v. light chain CDR2 having a sequence set forth in SEQ ID
NO: 29; and [0041] vi. light chain CDR3 having a sequence set forth
in SEQ ID NO: 30.
[0042] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises heavy chain variable region
set forth in SEQ ID NO: 39, or an analog or derivative thereof
having at least 90% sequence identity with the heavy chain variable
region sequence.
[0043] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises light chain variable region
set forth in SEQ ID NO: 40, or an analog thereof having at least
90% sequence identity with the light chain variable region
sequence.
[0044] According to a specific embodiment, the isolated monoclonal
antibody or fragment thereof comprises a heavy chain variable
region having a sequence set forth in SEQ ID NO: 39, and a light
chain variable region having a sequence set forth in SEQ ID NO: 40,
or an analog thereof having at least 90% sequence identity with the
light and/or heavy chain sequence.
[0045] The invention also encompasses antibody or antibody fragment
capable of binding with high affinity to an epitope within the
human Nectin4 protein to which monoclonal antibody hNec4.11
binds.
[0046] According to some embodiments, the isolated monoclonal
antibody or fragment comprises the CDR sequences of a monoclonal
antibody denoted hNec4.01 (or Nectin4.01), namely, the three CDR
sequences contained in heavy chain variable region set forth in SEQ
ID NO: 35 and the three CDR sequences contained in light chain
variable region set forth in SEQ ID NO: 36.
[0047] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises heavy chain CDR1
comprising the sequence AYNIH (SEQ ID NO: 9). According to some
embodiments, the isolated monoclonal antibody or the antibody
fragment comprises heavy chain CDR2 comprising the sequence
YIYPNNGGSGYNQKFMN (SEQ ID NO: 10). According to some embodiments,
the isolated monoclonal antibody or the antibody fragment comprises
heavy chain CDR3 comprising the sequence FDYDEAWFIY (SEQ ID NO:
11).
[0048] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) HC CDR1 comprising
the sequence AYNIH (SEQ ID NO: 9); (ii) HC CDR2 comprising the
sequence: YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); and (iii) HC CDR3
comprising the sequence: FDYDEAWFIY (SEQ ID NO: 11).
[0049] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises light chain CDR1
comprising the sequence SASSSVSYMH (SEQ ID NO: 12). According to
some embodiments, the isolated monoclonal antibody or the antibody
fragment comprises light chain CDR2 comprising the sequence DTSKLAS
(SEQ ID NO: 13). According to some embodiments, the isolated
monoclonal antibody or the antibody fragment comprises light chain
CDR3 comprising the sequence FQGSGSPYT (SEQ ID NO: 14).
[0050] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) LC CDR1 comprising
the sequence SASSSVSYMH (SEQ ID NO: 12); (ii) LC CDR2 comprising
the sequence: DTSKLAS (SEQ ID NO: 13); and (iii) HC CDR3 comprising
the sequence: FQGSGSPYT (SEQ ID NO: 14).
[0051] According to some specific embodiments the isolated
monoclonal antibody or fragment comprises heavy chain CDR1 sequence
comprising the sequence: AYNIH (SEQ ID NO: 9), heavy chain CDR2
comprising the sequence: YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), heavy
chain CDR3 comprising the sequence: FDYDEAWFIY (SEQ ID NO: 11),
light chain CDR1 comprising the sequence: SASSSVSYMH (SEQ ID NO:
12), light chain CDR2 comprising the sequence: DTSKLAS (SEQ ID NO:
13), and light chain CDR3 comprising the sequence: FQGSGSPYT (SEQ
ID NO: 14), or analogs thereof comprising no more than 5% amino
acid substitution, deletion and/or insertion in the hypervariable
region (HVR) sequence.
[0052] According to some specific embodiments the isolated
monoclonal antibody or fragment comprises a set of six CDR
sequences consisting of: [0053] i. heavy chain CDR1 having a
sequence set forth in SEQ ID NO: 9; [0054] ii. heavy chain CDR2
having a sequence set forth in SEQ ID NO: 10; [0055] iii. heavy
chain CDR3 having a sequence set forth in SEQ ID NO: 11; [0056] iv.
light chain CDR1 having a sequence set forth in SEQ ID NO: 12;
[0057] v. light chain CDR2 having a sequence set forth in SEQ ID
NO: 13; and [0058] vi. light chain CDR3 having a sequence set forth
in SEQ ID NO: 14.
[0059] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises heavy chain variable region
set forth in SEQ ID NO: 35, or an analog or derivative thereof
having at least 90% sequence identity with the heavy chain variable
region sequence.
[0060] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises light chain variable region
set forth in SEQ ID NO: 36, or an analog thereof having at least
90% sequence identity with the light chain variable region
sequence.
[0061] According to a specific embodiment, the isolated monoclonal
antibody or fragment thereof comprises a heavy chain variable
region having a sequence set forth in SEQ ID NO: 35, and a light
chain variable region having a sequence set forth in SEQ ID NO: 36,
or an analog thereof having at least 90% sequence identity with the
light and/or heavy chain sequence.
[0062] The invention also encompasses antibody or antibody fragment
capable of binding with high affinity to an epitope within the
human Nectin4 protein to which monoclonal antibody hNec4.01
binds.
[0063] According to some embodiments, the isolated monoclonal
antibody or fragment comprises the CDR sequences of a monoclonal
antibody denoted hNec4.05 (or Nectin4.05), namely, the three CDR
sequences contained in heavy chain variable region set forth in SEQ
ID NO: 37 and the three CDR sequences contained in light chain
variable region set forth in SEQ ID NO: 38.
[0064] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises heavy chain CDR1
comprising the sequence TYYIH (SEQ ID NO: 15). According to some
embodiments, the isolated monoclonal antibody or the antibody
fragment comprises heavy chain CDR2 comprising the sequence
WIYPGNVNTKNNEKFKV (SEQ ID NO: 16). According to some embodiments,
the isolated monoclonal antibody or the antibody fragment comprises
heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO:
17).
[0065] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) HC CDR1 comprising
the sequence TYYIH (SEQ ID NO: 15); (ii) HC CDR2 comprising the
sequence: WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); and (iii) HC CDR3
comprising the sequence: SNPYVMDY (SEQ ID NO: 17).
[0066] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises light chain CDR1
comprising the sequence KASQSVSNDVA (SEQ ID NO: 18). According to
some embodiments, the isolated monoclonal antibody or the antibody
fragment comprises light chain CDR2 comprising the sequence YASNRYT
(SEQ ID NO: 19). According to some embodiments, the isolated
monoclonal antibody or the antibody fragment comprises light chain
CDR3 comprising the sequence QQDYSSPYT (SEQ ID NO: 20).
[0067] According to certain embodiments, the isolated monoclonal
antibody or the antibody fragment comprises: (i) LC CDR1 comprising
the sequence KASQSVSNDVA (SEQ ID NO: 18); (ii) LC CDR2 comprising
the sequence: YASNRYT (SEQ ID NO: 19); and (iii) HC CDR3 comprising
the sequence: QQDYSSPYT (SEQ ID NO: 20).
[0068] According to some specific embodiments the isolated
monoclonal antibody or fragment comprises heavy chain CDR1 sequence
comprising the sequence: TYYIH (SEQ ID NO: 15), heavy chain CDR2
comprising the sequence: WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), heavy
chain CDR3 comprising the sequence: SNPYVMDY (SEQ ID NO: 17), light
chain CDR1 comprising the sequence: KASQSVSNDVA (SEQ ID NO: 18),
light chain CDR2 comprising the sequence: YASNRYT (SEQ ID NO: 19),
and light chain CDR3 comprising the sequence: QQDYSSPYT (SEQ ID NO:
20), or analogs thereof comprising no more than 5% amino acid
substitution, deletion and/or insertion in the hypervariable region
(HVR) sequence.
[0069] According to some specific embodiments the isolated
monoclonal antibody or fragment comprises a set of six CDR
sequences consisting of: [0070] i. heavy chain CDR1 having a
sequence set forth in SEQ ID NO: 15; [0071] ii. heavy chain CDR2
having a sequence set forth in SEQ ID NO: 16; [0072] iii. heavy
chain CDR3 having a sequence set forth in SEQ ID NO: 17; [0073] iv.
light chain CDR1 having a sequence set forth in SEQ ID NO: 18;
[0074] v. light chain CDR2 having a sequence set forth in SEQ ID
NO: 19; and [0075] vi. light chain CDR3 having a sequence set forth
in SEQ ID NO: 20.
[0076] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises heavy chain variable region
sequence set forth in SEQ ID NO: 37, or an analog or derivative
thereof having at least 90% sequence identity with the heavy chain
variable region sequence.
[0077] According to some embodiments, the isolated monoclonal
antibody or fragment thereof comprises light chain variable region
set forth in SEQ ID NO: 38, or an analog thereof having at least
90% sequence identity with the light chain variable region
sequence.
[0078] According to a specific embodiment, the isolated monoclonal
antibody or fragment thereof comprises a heavy chain variable
region having a sequence set forth in SEQ ID NO: 37, and a light
chain variable region having a sequence set forth in SEQ ID NO: 38,
or an analog thereof having at least 90% sequence identity with the
light and/or heavy chain sequence.
[0079] The invention also encompasses antibody or antibody fragment
capable of binding with high affinity to an epitope within the
human Nectin4 protein to which monoclonal antibody hNec4.05
binds.
[0080] According to some embodiments, the isolated antibody or
fragment thereof recognizes human Nectin4 with an affinity of at
least 10.sup.-8M. According to other embodiments, the antibody or
antibody fragment binds human Nectin4 with an affinity of
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M or even higher. According to some
embodiments, the antibody or antibody fragment binds to human
Nectin4 with an affinity of 10.sup.-9M to 10.sup.-10M. Each
possibility represents a separate embodiment of the invention.
[0081] Analogs and derivatives of the isolated mAbs, and the
fragments described above, are also within the scope of the
invention.
[0082] According to some embodiments, the antibody or antibody
fragment analog have at least 90% sequence identity with the
hypervariable region of the reference antibody sequence.
[0083] According to certain embodiments, the analog or derivative
of the isolated antibody or fragment thereof has at least 91, 92,
93, 94, 95, 96, 97, 98 or 99% sequence identity with a variable
region of the reference antibody sequence. Each possibility
represents a separate embodiment of the invention.
[0084] According to some embodiments, the antibody or antibody
fragment according to the invention comprises a heavy chain
variable region set forth in SEQ ID NO: 39, SEQ ID NO: 35 or SEQ ID
NO: 37, or an analog having at least 95% sequence similarity with
said sequence.
[0085] According to some embodiments, the antibody or antibody
fragment comprises a light chain variable region set forth in SEQ
ID NO: 40, SEQ ID NO: 36 or SEQ ID NO: 38, or an analog having at
least 95% sequence similarity with said sequence.
[0086] According to some embodiments, the antibody or antibody
fragment comprises a heavy chain and a light chain, wherein: (i)
the heavy chain comprises SEQ ID NO: 39 and the light chain
comprises SEQ ID NO: 40; (ii) the heavy chain comprises SEQ ID NO:
35 and the light chain comprises SEQ ID NO: 36; or (iii) the heavy
chain comprises SEQ ID NO: 37 and the light chain comprises SEQ ID
NO: 38. Analogs of the antibodies or fragments, having at least 95%
sequence similarity with said heavy or light chains are also
included.
[0087] According to some embodiments, the analog has at least 96,
97, 98 or 99% sequence identity with an antibody light or heavy
chain variable regions described above. According to some
embodiments, the analog comprises no more than one amino acid
substitution, deletion or addition to one or more CDR sequences of
the hypervariable region, namely, any one of the CDR sequences set
forth in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20. Each possibility represents a separate
embodiment of the present invention. According to some embodiments,
the amino acid substitution is a conservative substitution.
[0088] According to some embodiments, the antibody or antibody
fragment comprises a hypervariable region (HVR) having light and
heavy chain regions defined above, in which 1, 2, 3, 4, or 5 amino
acids were substituted, deleted and/or added. Each possibility
represents a separate embodiment of the invention.
[0089] According to some embodiments, the antibody or antibody
fragment comprises an HVR having light and heavy chain regions
defined above, in which one amino acid was substituted. According
to specific embodiments, the antibody or antibody fragment
comprises a CDR as defined above, in which one amino acid was
substituted.
[0090] According to some embodiments, the isolated monoclonal
antibody or the antibody fragment comprises a CDR set selected from
the group consisting of: [0091] i. a set of six CDRs wherein: HC
CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID
NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is
KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29);
and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30); [0092] ii. a set of six
CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC CDR2 is
YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY (SEQ ID
NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12); LC CDR2 is DTSKLAS
(SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and
[0093] iii. a set of six CDRs wherein: HC CDR1 sequence is TYYIH
(SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC
CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA (SEQ ID
NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is
QQDYSSPYT (SEQ ID NO: 20).
[0094] The present invention also provides monoclonal antibodies
and binding fragments thereof, comprising a heavy chain and a light
chain, wherein said chains comprises a set of heavy chain variable
region sequence and light chain variable region sequence, said set
is selected from the group consisting of: [0095] i. a set
comprising SEQ ID NOs: 39 and 40; [0096] ii. a set comprising SEQ
ID NOs: 35 and 36; and [0097] iii. a set comprising SEQ ID NOs: 37
and 38.
[0098] According to some embodiments, the antibody or antibody
fragment is capable of inhibiting human Nectin4 binding to TIGIT
expressed on T cells or NK cells.
[0099] According to a specific embodiment, the mAb is selected from
the group consisting of: chimeric antibody and an antibody fragment
comprising at least the antigen-binding portion of an antibody.
According to specific embodiments, the antibody is a chimeric
antibody. According to yet other embodiments, the chimeric antibody
comprised human constant region. According to a specific
embodiment, the antibody fragment is selected from the group
consisting of: Fab, Fab', F(ab').sub.2, Fd, Fd', Fv, dAb, isolated
CDR region, single chain variable region (scFV), single chain
antibody (scab), "diabodies", and "linear antibodies". Each
possibility represents a separate embodiment of the present
invention.
[0100] A single chain variable region (scFV) comprising the heavy
chain and light chain variable regions of the antibodies described
herein is also provided according to the present invention.
According to certain embodiments, there is a hinge region between
the variable regions.
[0101] According to some embodiments, the scFV sequence is set
forth in SEQ ID NO: 32, SEQ ID NO: 34, or an analog thereof having
at least 90% sequence similarity to said sequences.
[0102] According to some embodiments, the antibody comprises a
constant region sequence selected from the group consisting of:
mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human
IgG2, human IgG3 and human IgG4. Each possibility represents a
separate embodiment of the present invention.
[0103] According to some specific embodiments, the monoclonal
antibody is a chimeric monoclonal antibody.
[0104] According to some embodiments, the chimeric antibody
comprises human-derived constant regions.
[0105] According to some embodiments the human constant regions of
the chimeric antibody are selected from the group consisting of:
human IgG1, human IgG2, human IgG3, and human IgG4.
[0106] According to specific embodiments, the antibody is a human
IgG1. According to some embodiments, a human IgG comprising the
variable regions of the antibodies described herein is
provided.
[0107] According to some embodiments, a conjugate comprising an
antibody or fragment thereof as described above is provided.
[0108] According to some embodiments, the conjugate comprises a
carrier protein.
[0109] A chimeric antigen receptor (CAR) comprising an
extracellular portion (binding domain), capable of binding to
Nectin4 is provided according to another aspect of the present
invention.
[0110] According to some embodiments, the CAR comprises an
extracellular portion containing any of the provided antibodies or
fragment thereof as described herein.
[0111] According to some embodiments, the CAR comprises a Nectin4
binding site comprising a CDR set selected from the group
consisting of: [0112] i. a set of six CDRs wherein: HC CDR1 is
SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO:
26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA
(SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is
QQAYRSPYT (SEQ ID NO: 30); [0113] ii. a set of six CDRs wherein: HC
CDR1 is AYNIH (SEQ ID NO: 9); HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID
NO: 10); HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11); LC CDR1 is
SASSSVSYMH (SEQ ID NO: 12); LC CDR2 is DTSKLAS (SEQ ID NO: 13); and
LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and [0114] iii. a set of six
CDRs wherein: HC CDR1 sequence is TYYIH (SEQ ID NO: 15); HC CDR2 is
WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC CDR3 is SNPYVMDY (SEQ ID NO:
17); LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18); LC CDR2 is YASNRYT
(SEQ ID NO: 19); and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).
[0115] According to some embodiments, the CAR comprises an antigen
binding domain comprising SEQ ID NOs: 32 or 34, a transmembrane
domain, and an intracellular T cell signaling domain.
[0116] According to an aspect, the present invention provides an
isolated nucleic acid molecule encoding a CAR comprising an
antibody or antibody fragment which includes Nectin4 binding domain
comprising a CDR set selected from the group consisting of: [0117]
i. a set of six CDRs wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC
CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ
ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is
YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);
[0118] ii. a set of six CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO:
9); HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is
FDYDEAWFIY (SEQ ID NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12);
LC CDR2 is DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ
ID NO: 14); and [0119] iii. a set of six CDRs wherein: HC CDR1
sequence is TYYIH (SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV
(SEQ ID NO: 16); HC CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is
KASQSVSNDVA (SEQ ID NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19);
and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).
[0120] According to some embodiments, a vector comprises a
polynucleotide sequence set forth in SEQ ID NO: 31 or SEQ ID NO:
33, or an analog having at least 95% similarity with said sequence
is provided.
[0121] According to some embodiments, a T cell engineered to
express the CAR described herein is provided.
[0122] According to additional embodiments, an NK cell engineered
to express the CAR described herein is provided.
[0123] Polynucleotide sequences encoding monoclonal antibodies,
having high affinity and specificity for human Nectin4, as well as
vectors and host cells carrying these polynucleotide sequences, are
provided according to another aspect of the present invention.
[0124] According to some embodiments, polynucleotide sequences
encoding the amino acid sequences of heavy chain variable region
and light chain variable region described above are provided.
[0125] According to some embodiments, the polynucleotide sequence
encodes an antibody or antibody fragment or chain capable of
binding to an epitope within the human Nectin4 protein to which
binds: (i) a monoclonal antibody (herein identified as hNec4.11)
having a heavy chain variable region of SEQ ID NO: 39 and a light
chain variable region of SEQ ID NO: 40; (ii) a monoclonal antibody
(herein identified as hNec4.01) having a heavy chain variable
region of SEQ ID NO: 35 and a light chain variable region of SEQ ID
NO: 36; or (iii) a monoclonal antibody (herein identified as
hNec4.05) having a heavy chain variable region of SEQ ID NO: 37 and
a light chain variable region of SEQ ID NO: 38.
[0126] According to some embodiments, the polynucleotide sequence
encodes an antibody or antibody fragment or chain comprising the
sequence set forth in a sequence selected from the group consisting
of: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 35 and SEQ ID NO:
36; or SEQ ID NO: 37 and SEQ ID NO: 38. Each possibility represents
a separate embodiment of the present invention.
[0127] According to yet some embodiments, the polynucleotide
sequence according to the invention encodes an antibody or antibody
fragment or chain comprising: [0128] i. a set of six CDRs wherein:
HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ
ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is
KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29);
and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30); [0129] ii. a set of six
CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC CDR2 is
YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY (SEQ ID
NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12); LC CDR2 is DTSKLAS
(SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and
[0130] iii. a set of six CDRs wherein: HC CDR1 sequence is TYYIH
(SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC
CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA (SEQ ID
NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is
QQDYSSPYT (SEQ ID NO: 20).
[0131] Each possibility represents a separate embodiment of the
present invention.
[0132] According to some embodiments, the polynucleotide sequences
defined above encode a molecule selected from the group consisting
of: an antibody, an antibody fragment comprising at least an
antigen-binding portion, and an antibody conjugate comprising said
antibody or antibody fragment. Each possibility represents a
separate embodiment of the present invention.
[0133] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region
comprises a sequence set forth in SEQ ID NO: 21 or a variant
thereof having at least 90% sequence identity.
[0134] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region
comprises a sequence set forth in SEQ ID NO: 1 or a variant thereof
having at least 90% sequence identity.
[0135] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region,
comprises a sequence set forth in SEQ ID NO: 5, or a variant
thereof having at least 90% sequence identity.
[0136] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 23 or a variant
thereof having at least 90% sequence identity.
[0137] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 3 or a variant thereof
having at least 90% sequence identity.
[0138] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 7, or a variant
thereof having at least 90% sequence identity.
[0139] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region
comprises a sequence set forth in SEQ ID NO: 45 or a variant
thereof having at least 90% sequence identity.
[0140] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region
comprises a sequence set forth in SEQ ID NO: 41 or a variant
thereof having at least 90% sequence identity.
[0141] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody heavy chain variable region,
comprises a sequence set forth in SEQ ID NO: 43, or a variant
thereof having at least 90% sequence identity.
[0142] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 46 or a variant
thereof having at least 90% sequence identity.
[0143] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 42 or a variant
thereof having at least 90% sequence identity.
[0144] According to some embodiments, the polynucleotide sequence
encoding a monoclonal antibody light chain variable region
comprises a sequence set forth in SEQ ID NO: 44, or a variant
thereof having at least 90% sequence identity.
[0145] The present invention provides, according to some
embodiments, a polypeptide comprising at least one sequence encoded
by at least one polynucleotide sequence disclosed above.
[0146] In a further aspect, the present invention provides a
nucleic acid construct comprising a nucleic acid molecule encoding
at least one antibody chain or fragment thereof according to the
present invention. According to some embodiments the nucleic acid
construct is a plasmid.
[0147] According to some embodiments the plasmid comprises at least
one polynucleotide sequence set forth in a sequence selected from
the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:
41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44. Each
possibility represents a separate embodiment of the present
invention.
[0148] In still another aspect the present invention provides a
cell capable of producing an antibody or an antibody fragment
comprising the specific CDR sequences and/or specific heavy and
light chain variable regions defined above.
[0149] According to some embodiments, a cell is provided comprising
at least one polynucleotide sequence disclosed above.
[0150] According to some embodiments, the cell is cable of
producing a monoclonal antibody comprising: [0151] i. a set of six
CDRs wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is
WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO:
27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT
(SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30); [0152]
ii. a set of six CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC
CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY
(SEQ ID NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12); LC CDR2 is
DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14);
and [0153] iii. a set of six CDRs wherein: HC CDR1 sequence is
TYYIH (SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO:
16); HC CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA
(SEQ ID NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is
QQDYSSPYT (SEQ ID NO: 20).
[0154] Each possibility represents a separate embodiment of the
present invention.
[0155] According to some embodiments, the cell producing the
monoclonal antibody in a hybridoma cell.
[0156] Antibodies or fragments thereof according to the present
invention may be attached to a cytotoxic moiety, a radioactive
moiety, or an identifiable moiety.
[0157] The present invention provides, according to another aspect,
a pharmaceutical composition comprising as an active ingredient, at
least one antibody, antibody fragment or conjugates thereof, that
recognizes human Nectin4 with high affinity and specificity, and
optionally at least one pharmaceutical acceptable excipient,
diluent, salt or carrier, wherein said at least one antibody or
antibody fragment is capable of inhibiting the binding of human
Nectin4 to human TIGIT.
[0158] According to some embodiments, the pharmaceutical
composition comprises a mAb specific to Nectin4, wherein the mAb is
not conjugated to any toxin or anti-tumor agent.
[0159] According to some embodiments, the pharmaceutical
composition comprises a monoclonal antibody or a fragment thereof
which is capable of binding to an epitope within the human Nectin4
protein to which binds a monoclonal antibody selected from the
group consisting of: hNec4.11, hNec4.01 and hNec4.05 having
variable region and CDR sequences disclosed above.
[0160] According to some embodiments, the pharmaceutical
composition comprises at least one monoclonal antibody comprising:
[0161] i. a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 25; HC
CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ ID NO: 27; LC CDR1 is SEQ ID
NO: 28; LC CDR2 is SEQ ID NO: 29; and LC CDR3 is SEQ ID NO: 30;
[0162] ii. a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 9; HC
CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID NO: 11; LC CDR1 is SEQ ID
NO: 12; LC CDR2 is SEQ ID NO: 13; and LC CDR3 is SEQ ID NO: 14; or
[0163] iii. a set of six CDRs wherein: HC CDR1 sequence is SEQ ID
NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3 is SEQ ID NO: 17; LC CDR1
is SEQ ID NO: 18; LC CDR2 is SEQ ID NO: 19; and LC CDR3 is SEQ ID
NO: 20.
[0164] Each possibility represents a separate embodiment of the
present invention.
[0165] According to some embodiments, the pharmaceutical
composition comprises a monoclonal antibody or fragment thereof
comprising a heavy chain variable region having a sequence selected
from the group consisting of SEQ ID NO: 39, SEQ ID NO: 35, and SEQ
ID NO: 37. Each possibility represents a separate embodiment of the
invention
[0166] According to some embodiments, the pharmaceutical
composition comprises a monoclonal antibody or fragment thereof
comprising a light chain variable region having a sequence selected
from the group consisting of SEQ ID NO: 40, SEQ ID NO: 36, and SEQ
ID NO: 38. Each possibility represents a separate embodiment of the
invention.
[0167] According to a specific embodiment, the pharmaceutical
composition comprises a monoclonal antibody or fragment thereof
comprising a heavy chain variable region having the sequence set
forth in SEQ ID NO: 39 and a light chain variable region having the
sequence set forth in SEQ ID NO: 40.
[0168] According to a specific embodiment, the pharmaceutical
composition comprises a monoclonal antibody or fragment thereof
comprising a heavy chain variable region having the sequence set
forth in SEQ ID NO: 35 and a light chain variable region having the
sequence set forth in SEQ ID NO: 36.
[0169] According to a specific embodiment, the pharmaceutical
composition comprises a monoclonal antibody or fragment thereof
comprising a heavy chain variable region having the sequence set
forth in SEQ ID NO: 37 and a light chain variable region having the
sequence set forth in SEQ ID NO: 38.
[0170] Also provided are pharmaceutical compositions, comprising at
least one antibody, antibody fragment or antibody conjugate
according to the invention, for use in restoring NK cytotoxicity by
inhibiting binding of Nectin4 to TIGIT expressed on NK cells.
[0171] According to some embodiments, the antibody, antibody
fragment or antibody conjugate is capable of inhibiting human
Nectin4 binding to TIGIT expressed on T-cells.
[0172] According to some embodiments, the pharmaceutical
composition according to the present invention is for use in cancer
immunotherapy or in enhancing immune response.
[0173] According to some embodiments, the pharmaceutical
composition further comprises human lymphocytes expressing
TIGIT.
[0174] According to some embodiments, the human lymphocytes are
killer cells selected from the group consisting of: T cells, NK
cells and natural killer T (NKT cells).
[0175] According to some embodiments, the killer cells are
autologous or allogenic.
[0176] According to some embodiments, the pharmaceutical
composition comprises autologous or allogenic NK cells expressing
TIGIT.
[0177] The cancer treatable with a composition according to the
present invention may be any cancer that expresses Nectin4.
According to some embodiments, the cancer overexpresses Nectin4.
According to some embodiments of the invention, the cancer is a
metastatic cancer. According to some embodiments, the
pharmaceutical composition according to the present invention is
for use in inhibiting formation or distribution of metastases or
reducing the total number of metastases in a subject.
[0178] According to some embodiments of the invention, the cancer
is selected from the group consisting of a melanoma, a breast
cancer, an ovarian cancer, a pancreatic cancer, a colorectal
cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung
cancer, a thyroid cancer, a prostate cancer, a brain cancer, a
renal cancer, a throat cancer, a laryngeal carcinoma, a bladder
cancer, a hepatic cancer, a fibrosarcoma, an endometrial cells
cancer, a glioblastoma, sarcoma, a myeloid, a leukemia and a
lymphoma. Each possibility represents a separate embodiment of the
invention.
[0179] According to some embodiments, the cancer is a solid cancer.
According to some specific embodiments, the solid cancer is
selected from the group consisting breast cancer, lung cancer,
bladder cancer, pancreatic cancer and ovarian cancer.
[0180] According to other embodiments, the cancer is hematologic
cancer. According to some embodiments, the pharmaceutical
composition if for use of treating cancer, together with human
lymphocytes.
[0181] According to some embodiments, the human lymphocytes are
killer cells selected from the group consisting of: T cells, NK
cells and NKT cells.
[0182] According to some embodiments, the killer cells are
autologous or allogenic.
[0183] According to some embodiments, the killer cells are NK
cells.
[0184] According to some embodiments, the pharmaceutical
composition according to the present invention is for use in
preventing or treating a viral infection.
[0185] According to some embodiments, the pharmaceutical
composition is for use of preventing infection with measles
virus.
[0186] According to yet another aspect, the present invention
provides a method of inhibiting binding of human Nectin4 to TIGIT
by using a monoclonal antibody or antibody fragment defined
above.
[0187] According to yet another aspect, the present invention
provides a method of treating cancer comprising administering to a
subject in need thereof, an antibody or fragment thereof which
binds to Nectin4 wherein the antibody of fragment thereof is not
conjugated to any toxin or anti-tumor agent.
[0188] According to an additional aspect, the present invention
provides a method for enhancing immune response in a subject in
need thereof comprising administering to said subject a
therapeutically effective amount of a monoclonal antibody, antibody
fragment or antibody conjugate defined above.
[0189] According to yet another aspect, the present invention
provides a method of treating cancer comprising administering to a
subject in need thereof, a pharmaceutical composition comprising a
therapeutically effective amount at least one antibody or antibody
fragment thereof, that recognizes human Nectin4 with high affinity
and specificity and capable of inhibiting its binding to its ligand
TIGIT.
[0190] According to some embodiments of the invention, the
therapeutically effective amount results in a decrease in tumor
size or in the number of metastases in the subject.
[0191] According to some embodiments, the method comprises
administering a pharmaceutical composition comprising a mAb that is
not conjugated to any toxin or anti-tumor agent.
[0192] According to some embodiments, the method of treating cancer
comprises administering or performing at least one additional
anti-cancer therapy. According to certain embodiments, the
additional anticancer therapy is surgery, chemotherapy,
radiotherapy, or immunotherapy.
[0193] According to some embodiments, the method of treating cancer
comprises administration of a monoclonal antibody that recognizes
human Nectin4 with high affinity and specificity and an additional
anti-cancer agent. According to some embodiments, the additional
anti-cancer agent is selected from the group consisting of:
immune-modulator, activated lymphocyte cell, kinase inhibitor and
chemotherapeutic agent.
[0194] According to other embodiments, the additional
immune-modulator is an antibody, antibody fragment or antibody
conjugate that binds to an antigen other than human Nectin4.
[0195] According to some embodiments, the additional
immune-modulator is an antibody against an immune checkpoint
molecule. According to some embodiments, the additional immune
modulator is an antibody against an immune checkpoint molecule
selected from the group consisting of human programmed cell death
protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related
cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3
(LAG3), CD137, OX40 (also referred to as CD134), killer cell
immunoglobulin-like receptors (KIR), TIGIT, PVR, CTLA-4, NKG2A,
GITR and any other checkpoint molecule or a combination thereof.
Each possibility represents a separate embodiment of the
invention.
[0196] According to some embodiments, the anti-cancer agent is
selected from the group consisting of: Erbitux, cytarabine,
fludarabine, fluorouracil, mercaptopurine, methotrexate,
thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine,
carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin,
carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa,
dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin,
idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide,
teniposide and any combination thereof. Each possibility represents
a separate embodiment of the invention.
[0197] According to some embodiments, the anti-cancer agent is
epidermal growth factor receptor (EGFR) inhibitor. According to
some embodiments, the EGFR inhibitor is selected from the group
consisting of: Cetuximab (Erbitux.RTM.), Panitumumab
(Vectibix.RTM.), and necitumumab (Portrazza.RTM.). According to
some embodiments, the EGFR inhibitor is Cetuximab
(Erbitux.RTM.).
[0198] According to some embodiments of the invention, the subject
is a human subject.
[0199] According to some embodiments of the invention, the use
further comprises the use of an agent that downregulates the
activity or expression of an immune co-inhibitory receptor.
[0200] According to some embodiments of the invention, the immune
cell is a T cell.
[0201] According to some embodiments of the invention, the immune
co-inhibitory receptor is selected from the group consisting of
PD-1, TIGIT, PVR, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96,
BY55, LAIR1, SIGLEC10, and 2B4. Each possibility represents a
separate embodiment of the invention.
[0202] According to an aspect, the present invention provides a
method for modulating immune system function and/or activity
comprising modulating the binding of Nectin4 to TIGIT using an
antibody according to the invention.
[0203] According to some embodiments, the method of treating cancer
involves preventing or reducing formation, growth or spread of
metastases in a subject.
[0204] According to some embodiments, the method of treating cancer
comprises administering to a subject in need thereof a
pharmaceutical composition comprising a mAb or antibody fragment
thereof, capable of inhibiting the binding of human Nectin4 to
human TIGIT, and further administrating to said subject human
lymphocytes.
[0205] According to some embodiments, the human lymphocytes are
killer cells selected from the group consisting of: T cells, NK
cells and NKT cells.
[0206] According to some embodiments, the killer cells are
autologous or allogenic.
[0207] According to some embodiments, the killer cells are NK
cells.
[0208] The present invention also provides a method of preventing
or treating a viral infection comprising administering to a subject
at least one mAb specific to human Nectin4, or a fragment thereof
comprising at least the antigen binding domain, wherein said mAb or
fragment thereof is capable of inhibiting binding of Nectin4 to
TIGIT.
[0209] According to some embodiments, a method of preventing
infection with measles virus is provided comprising administering a
mAb specific to human Nectin4, or a fragment thereof comprising at
least the antigen binding domain, wherein said mAb or fragment
thereof is capable of inhibiting binding of measles virus to human
Nectin4 expressed on epithelial cells. According to some
embodiments, the cells are epithelial cells. According to an
aspect, the present invention provides a method of diagnosing or
prognosing cancer or infectious disease in a subject, the method
comprises determining the expression level of Nectin4 in a
biological sample of said subject using at least one antibody as
described herein.
[0210] According to yet another aspect, the present invention
provides a method of treating cancer comprising administering to a
subject in need thereof, a therapeutically effective amount of a
cell comprising a CAR molecule as described herein.
[0211] The present invention further comprises, according to
another aspect, a method of determining or quantifying the
expression of Nectin4, the method comprising contacting a
biological sample with an antibody or antibody fragment, and
measuring the level of complex formation, wherein the antibody or
antibody fragment comprises: [0212] i. a set of six CDRs wherein:
HC CDR1 is SEQ ID NO: 25; HC CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ
ID NO: 27; LC CDR1 is SEQ ID NO: 28; LC CDR2 is SEQ ID NO: 29; and
LC CDR3 is SEQ ID NO: 30; [0213] ii. a set of six CDRs wherein: HC
CDR1 is SEQ ID NO: 9; HC CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID
NO: 11; LC CDR1 is SEQ ID NO: 12; LC CDR2 is SEQ ID NO: 13; and LC
CDR3 is SEQ ID NO: 14; or [0214] iii. a set of six CDRs wherein: HC
CDR1 sequence is SEQ ID NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3
is SEQ ID NO: 17; LC CDR1 is SEQ ID NO: 18; LC CDR2 is SEQ ID NO:
19; and LC CDR3 is SEQ ID NO: 20.
[0215] Determining and quantifying methods may be performed
in-vitro or ex-vivo according to some embodiments or may be used in
diagnosing conditions associated with expression of Nectin4. The
antibodies according to the present invention may be also used to
configure screening methods. For example, an enzyme-linked
immunosorbent assay (ELISA), or a radioimmunoassay (RIA), as well
as method such as IHC or FACS, can be constructed for measuring
levels of secreted or cell-associated polypeptide using the
antibodies and methods known in the art.
[0216] According to some embodiments, the method for detecting or
quantifying the presence of Nectin4 expressed on cells or secreted
to a biological medium, comprises the steps of: [0217] i.
incubating a sample with an antibody specific to human Nectin4 or
an antibody fragment thereof comprising at least an antigen-binding
portion; [0218] ii. detecting the bound Nectin4 using a detectable
probe.
[0219] According to some embodiments, the method further comprises
the steps of: [0220] iii. comparing the amount of (ii) to a
standard curve obtained from a reference sample containing a known
amount of Nectin4; and [0221] iv. calculating the amount of the
Nectin4 in the sample from the standard curve.
[0222] According to some particular embodiments the sample is a
body fluid.
[0223] According to some embodiments, the method is performed
in-vitro or ex-vivo.
[0224] A kit for measuring the expression or presence of Nectin4 in
biological sample is also provided comprising at least one antibody
or antibody fragment according to the present invention. According
to some embodiments, the kit comprises an antibody or antibody
fragment comprising: [0225] i. a set of six CDRs wherein: HC CDR1
is SEQ ID NO: 25; HC CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ ID NO:
27; LC CDR1 is SEQ ID NO: 28; LC CDR2 is SEQ ID NO: 29; and LC CDR3
is SEQ ID NO: 30; [0226] ii. a set of six CDRs wherein: HC CDR1 is
SEQ ID NO: 9; HC CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID NO: 11;
LC CDR1 is SEQ ID NO: 12; LC CDR2 is SEQ ID NO: 13; and LC CDR3 is
SEQ ID NO: 14; or [0227] iii. a set of six CDRs wherein: HC CDR1
sequence is SEQ ID NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3 is SEQ
ID NO: 17; LC CDR1 is SEQ ID NO: 18; LC CDR2 is SEQ ID NO: 19; and
LC CDR3 is SEQ ID NO: 20.
[0228] According to an aspect, the present invention provides a kit
for detecting cancer, the diagnostic kit comprises an antibody or
antibody fragment thereof as disclosed herein.
[0229] According to some embodiments, the invention provides a
method of diagnosing, assessing the severity or staging an
immune-related disease or a proliferative disease comprising
determining the expression, concentration or activity of Nectin4 in
a sample from a subject using an antibody according to the present
invention or a fragment or conjugate thereof, and comparing the
expression or activity of Nectin4 to a reference amount of Nectin4
expression, concentration or activity. Said reference amount may be
obtained from a sample taken from a normal subject, from the same
subject while being in a different stage of the disease or is
determined from clinical data of a large population of
subjects.
[0230] Further embodiments and the full scope of applicability of
the present invention will become apparent from the detailed
description given hereinafter. However, it should be understood
that the detailed description and specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0231] FIG. 1 A schematic illustration of the receptors involved in
TIGIT signaling on immune (NK) and tumor cells. Anti nectin4 Ab is
depicted.
[0232] FIG. 2 Antibody clones hNec4.05 and hNec4.01 show the best
blocking capability of TIGIT-Nectin4 interactions. The graph
depicts the Mean Fluorescence Intensity (MFI) values of FACS
staining of RAJI Burkitt's lymphoma cells transfected with Nectin4.
The cells were incubated with 0.2 .mu.g of different clones (as
indicated) and then incubated with 3 .mu.g of TIGIT-Ig followed by
staining with an anti-human secondary antibody.
[0233] FIG. 3 Quantification of the binding affinity of antibody
clones to Nectin4. The binding of fluorophore-labeled Nectin4-Ig
and the antibody clones hNec4.05 and clone hNec4.01 observed with
microscale thermophoresis. Measurements were repeated with at least
three independent protein preparations and average of results are
shown (.+-.SEM).
[0234] FIGS. 4A and 4B Antibody clones hNec4.05 and hNec4.01 block
Nectin4 and increase NK cytotoxicity. [.sup.35S]
methionine-labelled (4A) RAJI Burkitt's lymphoma cells transfected
with Nectin4 (4B) LNCap prostate carcinoma cells (naturally
expressing Nectin4), were incubated with 1 .mu.g/well of either
mouse IgG1 as a control antibody, or anti-Nectin4 antibodies
hNec4.01 or hNec4.05. After 1 h the cells were supplemented with NK
cells and incubated for 5 hours. The average specific killing
(.+-.s.d.) in various Effector:Target (E:T) ratios of the NK:cancer
cells is plotted. * indicates significant effect (p<0.05) of the
hNec4.01 and hNec4.05 clones in comparison to the control antibody.
Figure shows one representative experiment out of three performed.
The same effect was determined with MCF-7, MDA-MB-453, SK-BR-3 and
T47D cells--all breast cancer cell lines.
[0235] FIGS. 5A-5C Antibody clones do not bind murine Nectin4. (A)
Western blots of RAJI cells transfected with murine Nectin4
(indicated as mNectin4) and detected with commercial anti-murine
Nectin4 mAb (Clone 356704 which doesn't work for flow cytometry).
Expression was compared to RAJI cells expressing empty vector
(indicated as Empty). Staining for hGAPDH was used as loading
control. (B-C) FACS staining of RAJI cells transfected with murine
Nectin4 (black line histograms). Cells were stained with 0.2 .mu.g
of (B) clone hNec4.01 or (C) clone hNec4.05.
[0236] FIGS. 6A-6C Blocking of Nectin4-Nectin1 interactions. (A-B)
FACS staining of RAJI cells transfected with human-Nectin4. Cells
were pre-incubated with 1 .mu.g of (A) clone hNec4.01, or (B) clone
hNec4.05 and then incubated with 3 .mu.g of Nectin1-Ig (black line
histograms). Staining without blocking appears as gray line. Grey
filled histograms are background control staining of secondary
antibody only. (C) Mean Fluorescence Intensity (MFI) values of FACS
staining of A and B.
[0237] FIGS. 7A-7C In vivo effect of anti-Nectin4 mAb. (A)
SCID-beige mice were subcutaneously implanted with 5.times.10.sup.6
Raji cells that were either transfected with empty vector (EV) or
over expressed (OE) nectin4, alone (left panel, no NK) or together
with 1.times.10.sup.6 NK cells (right panel, NK). (B) SCID-beige
mice were subcutaneously implanted with 5.times.10.sup.6 Raji cells
over expressing nectin4 together with either 1.times.10.sup.6 NK
cells. Mice were treated with 75 ug of either a control Ab or with
anti Nectin4 clone hNec4.05 mAb, twice a week by intraperitoneal
injection. (C) SCID-beige mice were subcutaneously implanted with
5.times.10.sup.6 MDA-MB-453 cells, alone or with 7.times.10.sup.5
NK cells. Mice were then treated as in (B). Tumors were harvested
and weighed at day 21 (A), 27 (B), or 23 (C) post tumor injection.
N=7 for all mice experimental groups. (*) p<0.05.
[0238] FIGS. 8A-8C Antibody clones hNec4.05 and hNec4.11 binding to
cell surface Nectin-4. (A) Antibody binding to Nectin-4 expressed
on the human cell line MDA-MD-453 was evaluated by FACS analysis.
Shown are the EC50 values that were calculated following titration
of Ab binding (range of 20-0.01 nM), and the maximal binding signal
for each clone. Of note, similar results were seen with a chimeric
version of the Abs in which the murine IgG1 Fc was replaced with
human Fc. (B) Antibody binding to CHO cells transfected with
Cynomolgus (Cyno)-Nectin-4 and analyzed as in A. (C) Antibody
binding to CHO cells transfected with murine-Nectin-4 and analyzed
as in A. ND--not detected.
[0239] FIGS. 9A-9D Antibody clones hNec4.05 and hNec4.11 block the
binding of Nectin-4 to its ligands TIGIT and Nectin-1. Binding of
Nectin-4 ligands was assessed by FACS analysis. CHO cells
transfected with human-Nectin-4 were incubated with either human
TIGIT-Ig (A & C) or with human Nectin-1-Ig (B & D), both at
20 ug/ml, with or without anti-Nectin-4 clone hNec4.05 (A & B)
or clone hNec4.11 (C & D), both at 8 ug/ml. Robust binding
inhibition by the anti-Nectin-4 Abs is seen in all cases. Remaining
signal is likely due to ligand binding to other receptors expressed
by CHO cells, such as PVR, which are not affected by the
anti-Nectin-4 Abs.
[0240] FIGS. 10A-10B Human IgG1 chimeric Ab clones hNec4.05 and
hNec4.11 enhance NK cell activation in the presence of tumor cells.
Human NK cells (effector, E) were incubated with the target cells
(T) HT1376 (A) and MDA-MD-453 (B) at E:T ratio of 2:1. Incubation
was done in the presence of 12 ug/ml chimeric clones hNec4.05 and
hNec4.11 or a control hIgG1. After two hours, NK cells were assayed
for their degranulation and activation status by FACS analysis of
CD107a expression. Degranulation of NK cells in presence of control
hIgG1 was set as 1 and fold induction was calculated accordingly.
Shown are averages of 2-3 repeats and their normalized SDs.
[0241] FIGS. 11A-11D CAR-T driven by hNec4.11 leads to specific T
cell activation in the presence of tumor cells that express
Nectin-4. (A) Schematic drawing of the CART construct. (B) Jurkat
cells were transduced by lenti particles encoding for the construct
and GFP. The transduction efficacy was above 99% as judged by FACS
analysis of GFP expression. (C) Parental Jurkat cells or Jurkat
cells expressing the CART construct (Jurkat pHAGE2.4.11) were
incubated with the target cells HT1376 or MDA-MD-453 (MDA-453) for
48 hours after which media was collected and tested for IL-2
concentration as a way to assess Jurkat activation. The secretion
of IL-2 was significantly induced by the CART expression (*=0.003,
**=0.00014; two tailed student test). Shown is a representative
experiment of the two performed. (D) PBMCs from healthy donor were
transduced using the CART construct. The CART PBMCs were incubated
with HT1376 cells through a range of E:Ts (indicated at the X
axis). Killing of the target cells was significant where marked by
asterisk (***p<7*10.sup.-5).
DETAILED DESCRIPTION OF THE INVENTION
[0242] The present invention provides effective monoclonal
antibodies specific to the human Nectin4. The invention also
provides production and use of the mAbs as therapeutic agents. In
particular, the mAbs of the present invention may be used for
restoring and augmenting anti-tumor killing activity of immune
cells, and as diagnostic reagents.
[0243] While prior publication shows the use of an anti Nectin4 mAb
for targeting a drug to tumor cells overexpressing Nectin4, the
present invention discloses for the first time monoclonal
antibodies that directly potentiate the immune system against tumor
cells, by inhibiting the binding to Nectin4 to the inhibitory
receptor TIGIT of immune cells such as NK cells.
[0244] The antibodies of the present invention overcome the
disadvantages of antibodies specific to TIGIT, which are currently
tested for treatment of cancer. Anti-TIGIT antibodies may,
allegedly, skew the entire immune system towards activation by
blocking all immune cells expressing TIGIT receptor and cause,
potentially, autoimmune effects, while the anti-Nectin4 antibodies
of the present invention target only Nectin4 expressing cells which
are known to be overexpressed in tumors.
[0245] Furthermore, some of the antibodies of the present invention
may also lead to an immune independent killing of tumor cells,
potentially via their ability to block nectin4 interactions with
nectin1.
[0246] The term "antigen" as used herein refers to a molecule or a
portion of a molecule capable of eliciting antibody formation and
being specifically bound by an antibody. An antigen may have one or
more than one epitope. The specific binding referred to above is
meant to indicate that the antigen will react, in a highly
selective manner, with its corresponding antibody and not with the
multitude of other antibodies which may be evoked by other
antigens. An antigen according to some embodiments of the present
invention is a Nectin4 protein.
[0247] The term "Nectin4" or "Nectin Cell Adhesion Molecule 4", as
used herein refers to a single-pass type I membrane protein of 510
amino acids and a molecular mass of 55454 Da, also known as PVRL4;
LNIR; PRR4; and EDSS1. The Nectin4 protein contains two
immunoglobulin-like (Ig-like) C2-type domains and one Ig-like
V-type domain. It is involved in cell adhesion through
trans-homophilic and -heterophilic interactions. The soluble form
is produced by proteolytic cleavage at the cell surface by the
metalloproteinase ADAM17/TACE and the secreted form is found in
both breast tumor cell lines and breast tumor patients. An
exemplary Nectin4 according to the invention is set forth in
SwissPort, UniPort and GenBank symbols or accession numbers:
Q96NY8-NECT4_HUMAN; Q96NY8; B4DQW3; Q96K15; Q96NY8-1; Q96NY8-2;
ENSP00000356991; NP_112178.2; XP_005245565.1; XP_011508323.1;
XP_011508324.1; or XP_011508325.1.
[0248] The antibodies or a fragment thereof according to the
invention binds to an epitope in Nectin4. Specifically, the
antibodies bind to an epitope within the ectodomain (extracellular
part) of the Nectin4 protein sequence.
[0249] The term "antigenic determinant" or "epitope" as used herein
refers to the region of an antigen molecule that specifically
reacts with a particular antibody. Peptide sequences derived from
an epitope can be used, alone or in conjunction with a carrier
moiety, applying methods known in the art, to immunize animals and
to produce additional polyclonal or monoclonal antibodies. Isolated
peptides derived from an epitope may be used in diagnostic methods
to detect antibodies.
[0250] It should be noted that the affinity can be quantified using
known methods such as, Surface Plasmon Resonance (SPR) (described
in Scarano S, Mascini M, Turner A P, Minunni M. Surface plasmon
resonance imaging for affinity-based biosensors. Biosens
Bioelectron. 2010, 25: 957-66), and can be calculated using, e.g.,
a dissociation constant, Kd, such that a lower Kd reflects a higher
affinity.
[0251] Antibodies, or immunoglobulins, comprise two heavy chains
linked together by disulfide bonds and two light chains, each light
chain being linked to a respective heavy chain by disulfide bonds
in a "Y" shaped configuration. Proteolytic digestion of an antibody
yields Fv (Fragment variable) and Fc (Fragment crystalline)
domains. The antigen binding domains, Fab, include regions where
the polypeptide sequence varies. The term F(ab').sub.2 represents
two Fab' arms linked together by disulfide bonds. The central axis
of the antibody is termed the Fc fragment. Each heavy chain has at
one end a variable domain (V.sub.H) followed by a number of
constant domains (C.sub.H). Each light chain has a variable domain
(V.sub.L) at one end and a constant domain (CO at its other end,
the light chain variable domain being aligned with the variable
domain of the heavy chain and the light chain constant domain being
aligned with the first constant domain of the heavy chain (CH1).
The variable domains of each pair of light and heavy chains form
the antigen-binding site. The domains on the light and heavy chains
have the same general structure and each domain comprises four
framework regions, whose sequences are relatively conserved, joined
by three hyper-variable domains known as complementarity
determining regions (CDRs 1-3). These domains contribute
specificity and affinity of the antigen-binding site.
[0252] CDR identification or determination from a given heavy or
light chain variable sequence, is typically made using one of few
methods known in the art. For example, such determination is made
according to the Kabat (Wu T. T and Kabat E. A., J Exp Med, 1970;
132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003,
27:55-77).
[0253] When the term "CDR having a sequence", or a similar term is
used, it includes options wherein the CDR comprises the specified
sequences and also options wherein the CDR consists of the
specified sequence.
[0254] The antigen specificity of an antibody is based on the hyper
variable region (HVR), namely the unique CDR sequences of both
light and heavy chains that together form the antigen-binding
site.
[0255] The isotype of the heavy chain (gamma, alpha, delta, epsilon
or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,
respectively). The light chain is either of two isotypes (kappa,
.kappa. or lambda, .lamda.). Both isotopes are found in all
antibody classes.
[0256] The term "antibody" is used in the broadest sense and
includes monoclonal antibodies (including full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, and antibody fragments long enough to exhibit the
desired biological activity, namely binding to human Nectin4.
[0257] Antibody or antibodies according to the invention include
intact antibodies, such as polyclonal antibodies or monoclonal
antibodies (mAbs), as well as proteolytic fragments thereof, such
as the Fab or F(ab').sub.2 fragments. Single chain antibodies also
fall within the scope of the present invention.
Antibody Fragments
[0258] "Antibody fragments" comprise only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 1989, 341, 544-546) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab').sub.2 fragments, a bivalent
fragment including two Fab' fragments linked by a disulphide bridge
at the hinge region; (ix) single chain antibody molecules (e.g.
single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426;
and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85,
5879-5883); (x) "diabodies" with two antigen binding sites,
comprising a heavy chain variable domain (VH) connected to a light
chain variable domain (VL) in the same polypeptide chain (see,
e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies"
comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which,
together with complementary light chain polypeptides, form a pair
of antigen binding regions (Zapata et al. Protein Eng., 1995, 8,
1057-1062; and U.S. Pat. No. 5,641,870).
[0259] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from antibody
phage libraries. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (Carter et at, Bio/Technology 10:163-167 (1992)).
According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner. In other embodiments, the
antibody of choice is a single chain Fv fragment (scFv).
[0260] Single chain antibodies can be single chain composite
polypeptides having antigen binding capabilities and comprising
amino acid sequences homologous or analogous to the variable
regions of an immunoglobulin light and heavy chain i.e. linked
V.sub.H-V.sub.L or single chain Fv (scFv). Techniques for the
production of single-chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce single-chain antibodies to Nectin4.
[0261] The term "monoclonal antibody" (mAb) as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. mAbs may be
obtained by methods known to those skilled in the art. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., Nature 1975, 256, 495, or may be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal
antibodies may also be isolated from phage antibody libraries using
the techniques described, for example, in Clackson et al., Nature
1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991,
222:581-597.
[0262] The design and development of recombinant monovalent
antigen-binding molecules derived from monoclonal antibodies
through rapid identification and cloning of the functional variable
heavy (VH) and variable light (VL) genes and the design and cloning
of a synthetic DNA sequence optimized for expression in recombinant
bacteria are described in Fields et at. 2013, 8(6):1125-48.
[0263] The mAbs of the present invention may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, and IgD. A
hybridoma producing a mAb may be cultivated in-vitro or in-vivo.
High titers of mAbs can be obtained by in-vivo production where
cells from the individual hybridomas are injected
intra-peritoneally into pristine-primed Balb/c mice to produce
ascites fluid containing high concentrations of the desired mAbs.
mAbs may be purified from such ascites fluids, or from culture
supernatants, using methods well known to those of skill in the
art.
[0264] Anti-idiotype antibodies specifically immunoreactive with
the hypervariable regions of an antibody of the invention are also
comprehended.
[0265] The invention provides a monoclonal antibody or an antibody
fragment comprising an antigen binding domain (ABD) which comprises
three CDRs of a light chain and three CDRs of a heavy chain,
wherein said ABD has at least 90% sequence identity or similarity
with an ABD of a monoclonal mouse antibody comprising: (i) a heavy
variable chain comprising the amino acid sequence SEQ ID NO: 39 and
a light variable chain comprising the amino acid sequence SEQ ID
NO: 40 (herein identified as hNec4.11); (i) a heavy variable chain
comprising the amino acid sequence SEQ ID NO: 35 and a light
variable chain comprising the amino acid sequence SEQ ID NO: 36
(herein identified as hNec4.01); or (ii) a heavy variable chain
comprising the amino acid sequence SEQ ID NO: 37 and a light
variable chain comprising the amino acid sequence SEQ ID NO: 38
(herein identified as hNec4.05). Such antibody may have an ABD
domain having at least 93%, at least 94%, at least 95%, at least
96, at least 97, at least 98, at least 99% sequence identity or
similarity or 100% sequence identity with corresponding ABD of
antibodies hNec4.11, hNec4.01 or hNec4.05.
[0266] Sequence identity is the amount of amino acids or
nucleotides which match exactly between two different sequences.
Sequence similarity permits conservative substitution of amino
acids to be determined as identical amino acids.
[0267] The invention also provides conservative amino acid variants
of the antibody molecules according to the invention. Variants
according to the invention also may be made that conserve the
overall molecular structure of the encoded proteins. Given the
properties of the individual amino acids comprising the disclosed
protein products, some rational substitutions will be recognized by
the skilled worker. Amino acid substitutions, i.e., "conservative
substitutions," may be made, for instance, on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. The term "antibody analog" as used herein refers to an
antibody derived from another antibody by one or more conservative
amino acid substitutions.
[0268] The term "antibody variant" as used herein refers to any
molecule comprising the antibody of the present invention. For
example, fusion proteins in which the antibody or an
antigen-binding-fragment thereof is linked to another chemical
entity is considered an antibody variant.
[0269] Analogs and variants of the antibody sequences are also
within the scope of the present application. These include, but are
not limited to, conservative and non-conservative substitution,
insertion and deletion of amino acids within the sequence. Such
modification and the resultant antibody analog or variant are
within the scope of the present invention as long as they confer,
or even improve the binding of the antibody to the human
Nectin4.
[0270] Conservative substitutions of amino acids as known to those
skilled in the art are within the scope of the present invention.
Conservative amino acid substitutions include replacement of one
amino acid with another having the same type of functional group or
side chain, e.g., aliphatic, aromatic, positively charged,
negatively charged. These substitutions may enhance oral
bioavailability, penetration, and targeting to specific cell
populations, immunogenicity, and the like. One of skill will
recognize that individual substitutions, deletions or additions to
a peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. For example, according to one table known in the art, the
following six groups each contain amino acids that are conservative
substitutions for one another: [0271] 1) Alanine (A), Serine (S),
Threonine (T); [0272] 2) Aspartic acid (D), Glutamic acid (E);
[0273] 3) Asparagine (N), Glutamine (Q); [0274] 4) Arginine (R),
Lysine (K); [0275] 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and [0276] 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W).
[0277] It should be emphasized that the variant chain sequences are
determined by sequencing methods using specific primers. Different
sequencing methods employed on the same sequence may result in
slightly different sequences due to technical issues and different
primers, particularly in the sequence terminals Therefore,
different variants of the anti-Nectin4 variable chain sequences are
specified along the application.
[0278] The terms "molecule having the antigen-binding portion of an
antibody" and "antigen-binding-fragments" as used herein are
intended to include not only intact immunoglobulin molecules of any
isotype and generated by any animal cell line or microorganism, but
also the antigen-binding reactive fraction thereof, including, but
not limited to, the Fab fragment, the Fab' fragment, the
F(ab').sub.2 fragment, the variable portion of the heavy and/or
light chains thereof, Fab mini-antibodies (see e.g., WO 93/15210,
U.S. patent application Ser. No. 08/256,790, WO 96/13583, U.S.
patent application Ser. No. 08/817,788, WO 96/37621, U.S. patent
application Ser. No. 08/999,554), and single-chain antibodies
incorporating such reactive fraction, as well as any other type of
molecule in which such antibody reactive fraction has been
physically inserted. Such molecules may be provided by any known
technique, including, but not limited to, enzymatic cleavage,
peptide synthesis or recombinant techniques.
[0279] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species, or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)). In addition, complementarity
determining region (CDR) grafting may be performed to alter certain
properties of the antibody molecule including affinity or
specificity. A non-limiting example of CDR grafting is disclosed in
U.S. Pat. No. 5,225,539.
[0280] Chimeric antibodies are molecules of which different
portions are derived from different animal species, such as those
having a variable region derived from a murine mAb and a human
immunoglobulin constant region. Antibodies that have variable
region framework residues substantially from human antibody (termed
an acceptor antibody) and CDRs substantially from a mouse antibody
(termed a donor antibody) are also referred to as humanized
antibodies. Chimeric antibodies are primarily used to reduce
immunogenicity in application and to increase yields in production,
for example, where murine mAbs have higher yields from hybridomas
but higher immunogenicity in humans, such that human/murine
chimeric mAbs are used. Chimeric antibodies and methods for their
production are known in the art (for example PCT patent
applications WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and
U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and
5,225,539).
[0281] According to some specific embodiments, the monoclonal
antibody is a chimeric monoclonal antibody.
[0282] According to some embodiments, the chimeric antibody
comprises human-derived constant regions.
[0283] According to some embodiments the human constant regions of
the chimeric antibody are selected from the group consisting of:
human IgG1, human IgG2, human IgG3, and human IgG4.
[0284] According to a particular embodiment, a chimeric monoclonal
antibody which recognizes human Nectin4 is provided comprising:
[0285] i. a set of six CDRs wherein: HC CDR1 is (SEQ ID NO: 25); HC
CDR2 is (SEQ ID NO: 26); HC CDR3 is (SEQ ID NO: 27); LC CDR1 is
(SEQ ID NO: 28); LC CDR2 is (SEQ ID NO: 29); and LC CDR3 is (SEQ ID
NO: 30); [0286] ii. a set of six CDRs wherein: HC CDR1 is (SEQ ID
NO: 9); HC CDR2 is (SEQ ID NO: 10); HC CDR3 is (SEQ ID NO: 11); LC
CDR1 is (SEQ ID NO: 12); LC CDR2 is (SEQ ID NO: 13); and LC CDR3 is
(SEQ ID NO: 14); or [0287] iii. a set of six CDRs wherein: HC CDR1
sequence is (SEQ ID NO: 15); HC CDR2 is (SEQ ID NO: 16); HC CDR3 is
(SEQ ID NO: 17); LC CDR1 is (SEQ ID NO: 18); LC CDR2 is (SEQ ID NO:
19); and LC CDR3 is (SEQ ID NO: 20).
[0288] According to an aspect, the present invention provides a CAR
comprising an antibody fragment that bind specifically to
Nectin4.
[0289] According to some embodiments, the CAR comprises: i) a
specific binding agent that can specifically bind to nectin4; ii) a
spacer or hinge domain; iii) a trans-membrane domain (TM) that
anchor the CAR within the T cell membrane; iv) an endodomain which
transmits signals within the T cell.
[0290] According to some embodiments, the CAR comprises a CDR set
selected from the group consisting of: [0291] i. a set of six CDRs
wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is
WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO:
27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT
(SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30); [0292]
ii. a set of six CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC
CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY
(SEQ ID NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12); LC CDR2 is
DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14);
and iii. a set of six CDRs wherein: HC CDR1 sequence is TYYIH (SEQ
ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC CDR3
is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA (SEQ ID NO:
18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is QQDYSSPYT
(SEQ ID NO: 20).
[0293] According to an aspect, the present invention provides an
isolated nucleic acid molecule encoding a CAR comprising an
antibody or antibody fragment which includes Nectin4 binding domain
comprising a CDR set selected from the group consisting of: [0294]
i. a set of six CDRs wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC
CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ
ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is
YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);
[0295] ii. a set of six CDRs wherein: HC CDR1 is AYNIH (SEQ ID NO:
9); HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is
FDYDEAWFIY (SEQ ID NO: 11); LC CDR1 is SASSSVSYMH (SEQ ID NO: 12);
LC CDR2 is DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ
ID NO: 14); and [0296] iii. a set of six CDRs wherein: HC CDR1
sequence is TYYIH (SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV
(SEQ ID NO: 16); HC CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is
KASQSVSNDVA (SEQ ID NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19);
and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).
[0297] According to some embodiments, a vector comprises a
polynucleotide sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 33
is provided. According to certain embodiments the vector is a viral
vector. According to certain embodiments the viral vector is a
lentiviral vector.
[0298] According to some embodiments, a T cell engineered to
express the CAR described herein is provided.
[0299] With respect to the transmembrane domain, in various
embodiments, a CAR can be designed to comprise a transmembrane
domain that is attached to the extracellular domain of the CAR. A
transmembrane domain can include one or more additional amino acids
adjacent to the transmembrane region, e.g., one or more amino acid
associated with the extracellular region of the protein from which
the transmembrane was derived (e.g., 1, 2, 3, 4, 5, or more amino
acids of the extracellular region) and/or one or more additional
amino acids associated with the intracellular region of the protein
from which the transmembrane protein is derived (e.g., 1, 2, 3, 4,
5, or more amino acids of the intracellular region). According to
some embodiments, the transmembrane domain can be selected or
modified by amino acid substitution to avoid binding of such
domains to the transmembrane domains of the same or different
surface membrane proteins, e.g., to minimize interactions with
other members of the receptor complex.
[0300] According to some embodiments, the trans membrane domain is
a trans membrane domain of a protein selected from the group
consisting of the alpha, beta or zeta chain of the T-cell receptor,
CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Each
possibility represents a separate embodiment of the invention.
[0301] According to some embodiments, the antibody or antibody
fragment which includes Nectin4 binding domain is connected to the
transmembrane domain by a hinge region. According to some
embodiments, the hinge is from a human protein. According to some
embodiments, the hinge is a human Ig (immunoglobulin) hinge.
According to certain embodiments, the hinge is an IgG4 hinge or a
CD8a hinge.
[0302] The cytoplasmic domain or region of the CAR includes an
intracellular signaling domain. An intracellular signaling domain
is generally responsible for activation of at least one of the
normal effector functions of the immune cell in which the CAR has
been introduced. The term "effector function" refers to a
specialized function of a cell. Effector function of a T cell, for
example, may be cytolytic activity or helper activity including the
secretion of cytokines. Thus, the term "intracellular signaling
domain" refers to the portion of a protein which transduces the
effector function signal and directs the cell to perform a
specialized function. The term intracellular signaling domain is
meant to include any truncated portion of the intracellular
signaling domain sufficient to transduce the effector function
signal. Examples of intracellular signaling domains for use in the
CAR of the invention include the cytoplasmic sequences of the T
cell receptor (TCR) and co-receptors that act in concert to
initiate signal transduction following antigen receptor engagement,
as well as any derivative or variant of these sequences and any
recombinant sequence that has the same functional capability.
[0303] In some embodiments, signals generated through the TCR alone
are insufficient for full activation of the T cell and that a
secondary and/or costimulatory signal is also required. Thus, T
cell activation can be said to be mediated by two distinct classes
of cytoplasmic signaling sequences: those that initiate
antigen-dependent primary activation through the TCR (primary
intracellular signaling domains) and those that act in an
antigen-independent manner to provide a secondary or costimulatory
signal (secondary cytoplasmic domain, e g, a costimulatory
domain).
[0304] According to some embodiments, the intracellular signaling
domain is designed to comprise two or more, costimulatory signaling
domains According to some embodiments, the two or more
costimulatory signaling domains, are separated by a linker
molecule.
[0305] According to some embodiments, the linker molecule is a
glycine residue. According to some embodiments, the linker is an
alanine residue.
[0306] According to some embodiments, the CAR comprises a
costimulatory domain obtained from a protein selected from the
group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1
(CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Each possibility
represents a separate embodiment of the invention.
[0307] The invention also relates to nucleic acid molecules
encoding the CARs, together with vectors and host cells. The
invention also relates to a (heterologous) T cell comprising a CAR
of the invention, together with pharmaceutical compositions
comprising such CARs together with a suitable carrier or excipient.
The invention also relates to autologous T cell therapies
incorporating the T cells, compositions and CARs of the invention
(and corresponding medical uses).
[0308] According to some embodiments, the CAR-expressing cell
described herein can further comprise a second CAR, e.g., a second
CAR that includes a different antigen binding domain, e.g., to the
same target (Nectin4) or a different target. According to some
embodiments, when the CAR-expressing cell comprises two or more
different CARs, the antigen binding domains of the different CARs
can be such that the antigen binding domains do not interact with
one another.
[0309] According to some embodiments, a population of cells wherein
at least one cell in the population expresses a CAR having an
anti-Nectin4 domain described herein, and a second cell expressing
another agent, e.g., an agent which enhances the activity of a
CAR-expressing cell, is provided.
[0310] The invention also relates to a (heterologous) NK cell
comprising a CAR of the invention, together with pharmaceutical
compositions comprising such CARs together with a suitable carrier
or excipient. The invention also relates to autologous NK cell
therapies incorporating the NK cells, compositions and CARs of the
invention (and corresponding medical uses).
[0311] As used herein, "chimeric antigen receptor" or "CAR" refers
to an artificially constructed hybrid polypeptide comprising an
antigen-binding domain (e.g., an antigen-binding portion of an
antibody (e.g., a scFV)), a transmembrane domain, and a T-cell or
NK-cell signaling and/or T-cell or NK-cell activation domain CARs
have the ability to redirect T-cell or NK-cell specificity and
reactivity toward a selected target in a non-MHC-restricted manner,
exploiting the antigen-binding properties of monoclonal antibodies.
The non-MHC-restricted antigen recognition gives T-cells or
NK-cells expressing CARs the ability to recognize an antigen
independent of antigen processing, thus bypassing a major mechanism
of tumor escape. Most commonly, the CAR's extracellular binding
domain is composed of a single chain variable fragment (scFv)
derived from fusing the variable heavy and light regions of a
murine or humanized monoclonal antibody.
[0312] Pharmacology
[0313] In pharmaceutical and medicament formulations, the active
agent is preferably utilized together with one or more
pharmaceutically acceptable carrier(s) and optionally any other
therapeutic ingredients. The carrier(s) must be pharmaceutically
acceptable in the sense of being compatible with the other
ingredients of the formulation and not unduly deleterious to the
recipient thereof. The active agent is provided in an amount
effective to achieve the desired pharmacological effect, as
described above, and in a quantity appropriate to achieve the
desired exposure.
[0314] Typically, the antibodies and fragments and conjugates
thereof of the present invention comprising the antigen binding
portion of an antibody or comprising another polypeptide including
a peptide-mimetic will be suspended in a sterile saline solution
for therapeutic uses. The pharmaceutical compositions may
alternatively be formulated to control release of active ingredient
(molecule comprising the antigen binding portion of an antibody) or
to prolong its presence in a patient's system. Numerous suitable
drug delivery systems are known and include, e.g., implantable drug
release systems, hydrogels, hydroxymethylcellulose, microcapsules,
liposomes, microemulsions, microspheres, and the like. Controlled
release preparations can be prepared through the use of polymers to
complex or adsorb the molecule according to the present invention.
For example, biocompatible polymers include matrices of
poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride
copolymer of a stearic acid dimer and sebaric acid. The rate of
release of the molecule according to the present invention, i.e.,
of an antibody or antibody fragment, from such a matrix depends
upon the molecular weight of the molecule, the amount of the
molecule within the matrix, and the size of dispersed
particles.
[0315] The pharmaceutical composition of this invention may be
administered by any suitable means, such as orally, topically,
intranasally, subcutaneously, intramuscularly, intravenously,
intra-arterially, intraarticulary, intralesionally, intratumorally
or parenterally. Ordinarily, intravenous (i.v.) administration is
used for delivering antibodies.
[0316] It will be apparent to those of ordinary skill in the art
that the therapeutically effective amount of the molecule according
to the present invention will depend, inter alia upon the
administration schedule, the unit dose of molecule administered,
whether the molecule is administered in combination with other
therapeutic agents, the immune status and health of the patient,
the therapeutic activity of the molecule administered, its
persistence in the blood circulation, and the judgment of the
treating physician.
[0317] As used herein the term "therapeutically effective amount"
refers to an amount of a drug effective to treat a disease or
disorder in a mammal. In the case of cancer, the therapeutically
effective amount of the drug may reduce the number of cancer cells;
reduce the tumor size; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
disorder. To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic. For
cancer therapy, efficacy in vivo can, for example, be measured by
assessing the duration of survival, time to disease progression
(TTP), the response rates (RR), duration of response, and/or
quality of life.
[0318] The cancer amendable for treatment by the present invention
includes, but is not limited to: carcinoma, lymphoma, blastoma,
sarcoma, and leukemia or lymphoid malignancies. More particular
examples of such cancers include squamous cell cancer, lung cancer
(including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, and squamous carcinoma of the lung),
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer (including gastrointestinal cancer), pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast cancer, colon cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, liver cancer, prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma and various types of head
and neck cancer, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic
NHL; high-grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome. Preferably, the cancer is
selected from the group consisting of breast cancer, colorectal
cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins
lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer,
pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid
carcinoma, head and neck cancer, melanoma, ovarian cancer,
mesothelioma, and multiple myeloma. The cancerous conditions
amendable for treatment of the invention include metastatic
cancers.
[0319] According to other embodiments, the pharmaceutical
composition according to the invention are for use in treating
cancer characterized by overexpression of Nectin4. Nectin4
overexpression related cancer types can be identified using known
data bases such as The Cancer Genome Atlas (TCGA). According to
certain embodiments, the cancer treatable with a composition
according to the present invention is selected from the group
consisting of adrenocortical carcinoma (ACC), chromophobe renal
cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon
and rectal adenocarcinoma (COAD, READ), pancreatic ductal
adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG),
papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), head
and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma
(PRAD), uterine corpus endometrial carcinoma (UCEC), cervical
cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO),
urothelial bladder cancer (BLCA), clear cell kidney carcinoma
(KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma
(UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV),
papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM),
breast cancer (BRCA), lower grade glioma (LGG), and diffuse large
B-cell lymphoma (DLBC). Each possibility represents a separate
embodiment of the invention.
[0320] The molecules of the present invention as active ingredients
are dissolved, dispersed or admixed in an excipient that is
pharmaceutically acceptable and compatible with the active
ingredient as is well known. Suitable excipients are, for example,
water, saline, phosphate buffered saline (PBS), dextrose, glycerol,
ethanol, or the like and combinations thereof. Other suitable
carriers are well known to those skilled in the art. In addition,
if desired, the composition can contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents.
[0321] The pharmaceutical composition according to the present
invention may be administered together with an anti-neoplastic
composition.
[0322] The term "treatment" as used herein refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented.
[0323] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include melanoma, lung,
thyroid, breast, colon, prostate, hepatic, bladder, renal,
cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus,
sarcoma, biliary, or endometrial cancer.
[0324] According to some embodiments, the method of treating cancer
comprises administering the pharmaceutical composition as part of a
treatment regimen comprising administration of at least one
additional anti-cancer agent.
[0325] According to some embodiments, the anti-cancer agent is
selected from the group consisting of an antimetabolite, a mitotic
inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II
inhibitor, an asparaginase, an alkylating agent, an antitumor
antibiotic, and combinations thereof. Each possibility represents a
separate embodiment of the invention.
[0326] According to some embodiments, the antimetabolite is
selected from the group consisting of cytarabine, gludarabine,
fluorouracil, mercaptopurine, methotrexate, thioguanine,
gemcitabine, and hydroxyurea. According to some embodiments, the
mitotic inhibitor is selected from the group consisting of
vincristine, vinblastine, and vinorelbine. According to some
embodiments, the topoisomerase inhibitor is selected from the group
consisting of topotecan and irenotecan. According to some
embodiments, the alkylating agent is selected from the group
consisting of busulfan, carmustine, lomustine, chlorambucil,
cyclophosphamide, cisplatin, carboplatin, ifosamide,
mechlorethamine, melphalan, thiotepa, dacarbazine, and
procarbazine. According to some embodiments, the antitumor
antibiotic is selected from the group consisting of bleomycin,
dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin,
mitoxantrone, and plicamycin. According to some embodiments, the
topoisomerase II is selected from the group consisting of etoposide
and teniposide. Each possibility represents a separate embodiment
of the present invention.
[0327] According to some particular embodiments, the additional
anti-cancer agent is selected from the group consisting of
bevacizumab, carboplatin, cyclophosphamide, doxorubicin
hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride,
thiotepa, and combinations thereof. Each possibility represents a
separate embodiment of the present invention.
[0328] Monoclonal antibodies according to the present invention may
be used as part of combined therapy with at least one anti-cancer
agent. According to some embodiments, the additional anti-cancer
agent is an immuno-modulator, an activated lymphocyte cell, a
kinase inhibitor or a chemotherapeutic agent.
[0329] According to some embodiments, the anti-cancer agent is an
immuno-modulator, whether agonist or antagonist, such as antibody
against an immune checkpoint molecule.
[0330] Checkpoint immunotherapy blockade has proven to be an
exciting new venue of cancer treatment Immune checkpoint pathways
consist of a range of co-stimulatory and inhibitory molecules which
work in concert in order to maintain self-tolerance and protect
tissues from damage by the immune system under physiological
conditions. Tumors take advantage of certain checkpoint pathways in
order to evade the immune system. Therefore, the inhibition of such
pathways has emerged as a promising anti-cancer treatment
strategy.
[0331] The anti-cytotoxic T lymphocyte 4 (CTLA-4) antibody
ipilimumab (approved in 2011) was the first immunotherapeutic agent
that showed a benefit for the treatment of cancer patients. The
antibody interferes with inhibitory signals during antigen
presentation to T cells. Anti-programmed cell death 1 (PD-1)
antibody pembrolizumab (approved in 2014) blocks negative immune
regulatory signaling of the PD-1 receptor expressed by T cells. An
additional anti-PD-1 agent was filed for regulatory approval in
2014 for the treatment of non-small cell lung cancer (NSCLC).
Active research is currently exploring many other immune
checkpoints, among them: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA,
CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also
referred to as CD134), and killer cell immunoglobulin-like
receptors (KIR).
[0332] According to some specific embodiments, the immuno-modulator
is selected from the group consisting of: an antibody inhibiting
CTLA-4, an anti-human programmed cell death protein 1 (PD-1), PD-L1
and PD-L2 antibody, an activated cytotoxic lymphocyte cell, a
lymphocyte activating agent, an antibody against CEACAM, an
antibody against TIGIT, and a RAF/MEK pathway inhibitor. Each
possibility represents a separate embodiment of the present
invention. According to some specific embodiments, the additional
immuno-modulator is selected from mAb to PD-1, mAb to PD-L1, mAb to
PD-L2, mAb to CEACAM1, mAb to CTLA-4, mAB to TIGIT, PVR,
Interleukin 2 (IL-2) or lymphokine-activated killer (LAK) cell.
[0333] According to other embodiments the additional anti-cancer
agent is a chemotherapeutic agent. The chemotherapy agent, which
could be administered together with the antibody according to the
present invention, or separately, may comprise any such agent known
in the art exhibiting anticancer activity, including but not
limited to: mitoxantrone, topoisomerase inhibitors, spindle poison
vincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel,
docetaxel; alkylating agents: mechlorethamine, chlorambucil,
cyclophosphamide, melphalan, ifosfamide; methotrexate;
6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabin;
podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazin;
antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin;
nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin,
daunorubicin; inorganic ions: cisplatin, carboplatin; interferon,
asparaginase; hormones: tamoxifen, leuprolide, flutamide, and
megestrol acetate.
[0334] According to some embodiments, the chemotherapeutic agent is
selected from alkylating agents, antimetabolites, folic acid
analogs, pyrimidine analogs, purine analogs and related inhibitors,
vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase,
topoisomerase inhibitor, interferons, platinum coordination
complexes, anthracenedione substituted urea, methyl hydrazine
derivatives, adrenocortical suppressant, adrenocorticosteroides,
progestins, estrogens, antiestrogen, androgens, antiandrogen, and
gonadotropin-releasing hormone analog. According to another
embodiment, the chemotherapeutic agent is selected from the group
consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan,
oxaliplatin, capecitabine, paclitaxel and doxetaxel. One or more
chemotherapeutic agents can be used.
[0335] In some embodiments, the pharmaceutical composition
according to the present invention is for use in treating cancer or
for use in enhancing the immune response.
[0336] The term "enhancing immune response" refers to increasing
the responsiveness of the immune system and inducing or prolonging
its memory. The pharmaceutical composition according to the present
invention may be used to stimulate immune system upon vaccination.
Thus, in one embodiment the pharmaceutical composition can be used
for improving vaccination.
[0337] In certain embodiments, the cancer is selected from lung,
thyroid, breast, colon, melanoma, prostate, hepatic, bladder,
renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian,
uterus, sarcoma, biliary, and endometrial cells cancer. Each
possibility represents a separate embodiment of the invention.
[0338] According to some embodiments, a pharmaceutical composition,
comprising at least one antibody or fragment thereof according to
the present invention, and a pharmaceutical composition, comprising
an additional immuno-modulator or a kinase inhibitor, are used in
treatment of cancer by separate administration.
[0339] According to still another aspect the present invention
provides a method of treating cancer in a subject in need thereof
comprising administering to said subject a therapeutically
effective amount of a monoclonal antibody or antibody fragment
according to the present invention.
[0340] According to an additional aspect the present invention
provides methods for treating a disease associated with Nectin4
overexpression.
[0341] According to an aspect, the present invention provides a
method of treating cancer in a subject in need thereof comprising
administering to said subject a therapeutically effective amount of
a plurality of T-cells comprising a CAR molecule as described
herein.
[0342] The term "effective amount" as used herein refers to a
sufficient amount of the monoclonal antibody of the antibody
fragment that, when administered to a subject will have the
intended therapeutic effect. The effective amount required to
achieve the therapeutic end result may depend on a number of
factors including, for example, the specific type of the tumor and
the severity of the patient's condition, and whether the
combination is further co-administered with radiation. The
effective amount (dose) of the active agents, in the context of the
present invention should be sufficient to affect a beneficial
therapeutic response in the subject over time, including but not
limited to inhibition of tumor growth, reduction in the rate of
tumor growth, prevention of tumor and metastasis growth and
enhanced survival.
[0343] Toxicity and therapeutic efficacy of the compositions
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., by
determining the IC50 (the concentration which provides 50%
inhibition) and the maximal tolerated dose for a subject compound.
The data obtained from these cell culture assays and animal studies
can be used in formulating a range of dosages for use in humans.
The dosage may vary depending inter alia upon the dosage form
employed, the dosing regimen chosen, the composition of the agents
used for the treatment and the route of administration utilized,
among other relevant factors. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. Depending on the severity and
responsiveness of the condition to be treated, dosing can also be a
single administration of a slow release composition, with course of
treatment lasting from several days to several weeks or until cure
is affected or diminution of the disease state is achieved. The
amount of a composition to be administered will, of course, be
dependent on the subject being treated, the severity of the
affliction, the manner of administration, the judgment of the
prescribing physician, and all other relevant factors.
[0344] The term "administering" or "administration of" a substance,
a compound or an agent to a subject can be carried out using one of
a variety of methods known to those skilled in the art. For
example, a compound or an agent can be administered enterally or
parenterally, Enterally refers to administration via the
gastrointestinal tract including per us, sublingually or rectally.
Parenteral administration includes administration intravenously,
intradermally, intramuscularly, intraperitoneally, subcutaneously,
ocularly, sublingually, intranasally, by inhalation, intraspinally,
intracerebrally, and transdermally (by absorption, e.g., through a
skin duct). A compound or agent can also appropriately be
introduced by rechargeable or biodegradable polymeric devices or
other devices, e.g., patches and pumps, or formulations, which
provide for the extended, slow or controlled release of the
compound or agent.
[0345] Administering can also be performed, for example, once, a
plurality of times, and/or over one or more extended periods. In
some embodiments, the administration includes both direct
administration, including self-administration, and indirect
administration, including the act of prescribing a drug. For
example, as used herein, a physician who instructs a patient to
self-administer a drug, or to have the drug administered by another
and/or who provides a patient with a prescription for a drug is
administering the drug to the patient.
[0346] Antibodies are generally administered in the range of about
0.1 to about 20 mg/kg of patient weight, commonly about 0.5 to
about 10 mg/kg, and often about 1 to about 5 mg/kg. In this regard,
it is preferred to use antibodies having a circulating half-life of
at least 12 hours, preferably at least 4 days, more preferably up
to 21 days, Chimeric antibodies are expected to have circulatory
half-lives of up to 14-21 days. In some cases, it may be
advantageous to administer a large loading dose followed by
periodic (e.g., weekly) maintenance doses over the treatment
period. Antibodies can also be delivered by slow-release delivery
systems, pumps, and other known delivery systems for continuous
infusion.
[0347] The term "about" means that an acceptable error range, e.g.,
up to 5% or 10%, for the particular value should be assumed.
[0348] Diagnosis
[0349] The present invention further discloses methods for
diagnosing and prognosing cancer.
[0350] According to an aspect, the present invention provides a
diagnostic and/or prognostic method of cancer or infectious disease
in a subject, the method comprises the step of determining the
expression level of Nectin4 in a biological sample of said subject
using at least one antibody as described herein.
[0351] The term "biological sample" encompasses a variety of sample
types obtained from an organism that may be used in a diagnostic or
monitoring assay. The term encompasses blood and other liquid
samples of biological origin, solid tissue samples, such as a
biopsy specimen, or tissue cultures or cells derived there from and
the progeny thereof. Additionally, the term may encompass
circulating tumor or other cells. The term specifically encompasses
a clinical sample, and further includes cells in cell culture, cell
supernatants, cell lysates, serum, plasma, urine, amniotic fluid,
biological fluids including aqueous humour and vitreous for eyes
samples, and tissue samples. The term also encompasses samples that
have been manipulated in any way after procurement, such as
treatment with reagents, solubilisation, or enrichment for certain
components.
[0352] Determining the expression level of Nectin4 can be performed
by a labeled anti-Nectin4 antibody as described herein. Determining
the expression can be performed, for example, by ELISA. The method
of the invention can further comprise the step of comparing said
level of expression to a control level.
[0353] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed as limiting the scope of the invention.
EXAMPLES
Experimental Procedures
[0354] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non-limiting fashion.
[0355] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological, immunological and recombinant DNA
techniques. Such techniques are well known in the art. Other
general references referring to well-known procedures are provided
throughout this document for the convenience of the reader.
Methods
Cell Lines
[0356] The cell lines used were LNCap, JEG3, MCF-7, RAJI,
MDA-MD-453, HT1376 and CHO cells. In some cases, CHO cells were
stably transfected with cynomolgus (Cyno) or murine Nectin-4. The
cells were grown at 37.degree. C., >95% humidity and 5% CO.sub.2
in DMEM for all cells except RAJI cells, which were cultured in
RPMI supplemented with 10% heat inactivated FCS (media and sera
from Sigma-Aldrich).
Flow Cytometry
[0357] Flow cytometry was performed using anti murine Nectin4 mAb
(Clone 356704). Cells were incubated on ice for 30 minutes with 0.2
.mu.g of mAb per 100,000 cells. Detection was performed with a
secondary goat Ab coupled to AlexaFluor 647 (Jackson
ImmunoResearch) for 30 min on ice.
[0358] In some cases, anti-Nectin-4 Abs were used at various
concentrations and incubated on ice with target cells for 30
minutes. Detection was performed with secondary Abs directed
against murine or human Fc, coupled to AlexaFluor 647 (Jackson
ImmunoResearch) for 30 min on ice.
[0359] For staining of human TIGIT-Ig or human Nectin1-Ig, cells
were incubated on ice for 1 hour with 3 .mu.g of TIGIT-Ig per
100,000 cells. Detection was performed with a secondary Alexa Fluor
647 anti-human (Jackson ImmunoResearch) for 30 min on ice. For
blocking experiments cells were pre-incubated with 1 .mu.g of the
indicated antibody prior to the TIGIT-Ig or human Nectin1-Ig
staining. In some cases, cells were incubated with 8 ug/ml of the
indicated Ab together with human TIGIT-Ig or human Nectin-1-Ig (at
20 ug/ml). Detection of ligand binding was performed with a
secondary Alexa Fluor 647 anti-human Ab (Jackson
ImmunoResearch).
[0360] Analysis was performed using the FACS-Calibur (BD
Biosciences) or Cytoflex Becman Coulter flow cytometers and FCS
express software.
Killing Assay
[0361] For evaluation of NK cell cytotoxic activity against targets
cells, S.sup.35 release assays were performed as described
(Mandelboim et al., Exp. Med. 184(3):913-22). NK cells were
isolated from healthy donors using EasySep human NK separation kit
(19055 STEMCELL TECHNOLOGIES) and were grown with PHA and IL-2.
Target cells were incubated overnight in methionine free medium
with radioactive methionine [S.sup.35]. Next, the cells were washed
and incubated on ice with 1 .mu.g of the antibodies per 5000 cells
per well. The cells were then incubated with the NK cells for 5
hours. S.sup.35 release was measured with .beta.-counter TopCount
(Packard). The results are represented as:
(CPM(sample)-CPM(spontaneous release))/(CPM(total
release)-CPM(spontaneous release)).times.100, wherein CPM denotes
counts per minute.
ADCC Assay
[0362] NK cells were isolated from healthy donors using EasySep
human NK separation kit (19055 STEMCELL TECHNOLOGIES) and were
grown with PHA and IL-2. Target cells were plated at 2.5*10.sup.4
cells per well in 96U plates and co-incubated with activated NK
cells at E:T ratio of 2:1. Incubation was done in the presence of
12 ug/ml chimeric clones hNec4.05hIgG1 and hNec4.11hIgG1 or a
control hIgG1. After two hours NK cells were analyzed by FACS for
their CD107a (Biolegend cat 328619) degranulation marker
expression.
CAR-T Generation and Functional Assay
[0363] Single chain of the hNec4.11 Ab was cloned in-frame to the
CD8 stalk region followed by the CD28 TM domain, 41BB intracellular
domain, and the intracellular domain of CD3Zeta chain. A schematic
drawing of the CAR-T construct is shown in FIG. 11A. The construct
was introduced into a hEf1a containing promoter lentiviral vector
(pHAGE2) followed by IRES GFP cassette to monitor transduction
efficacy. Jurkat cells were transduced by lenti particles encoding
for the construct. The transduction efficacy was above 99% (FIG.
11B).
[0364] Parental Jurkat cells or Jurkat cells expressing the CAR-T
construct (Jurkat pHAGE2.4.11) (5*10.sup.4 per well) were incubated
with the target cells HT1376 and MDA-MD-453 at E:T ratio of 1:1 for
48 hours. Sups were collected after centrifugation and IL-2 levels
were measured using the IL-2 ELISA kit of Peprotech (cat 900-T12)
according to the manufacturer protocol.
[0365] PBMCs from healthy donor were preactivated for 72 hours,
using ImmunoCult.TM. Human CD3/CD28 T Cell Activator according to
the manufacturer protocol. The cells were transduced using
pHAGE2.4.11 lenti according to Kochenderfer J N et al. (J
Immunother. 2009 doi: 10.1097/CJI.0b013e3181ac6138) and the
expression was validated by GFP levels. The CART PBMCs were
incubated with HT1376 (2.5*10.sup.4/well) cells through a range of
E:Ts. After 48 hours the effector cells were removed, the target
cells were washed three times and viability measured using
CellTiter-Glo.RTM. Luminescent Cell Viability Assay according to
the manufacturer protocol.
In Vivo Mice Tumor Model
[0366] All experiments were performed using 6-8 weeks old
SCID-beige female mice. All mice were housed under SPF conditions,
normal light/dark cycles and 22+/-2.degree. C. in a specific
pathogen free unit of the Hebrew University Medical School
(Ein-Kerem, Jerusalem) and in accordance with the guidelines of the
ethics committee. Every group of mice contained 7 females (n=7).
Xenografts were generated by administering subcutaneous injection
of the indicated cells into the left flank region. Injection of
anti Nectin4 clone.05 and control (anti-murine CD3, InVivoMAb-clone
17A2) antibodies was administrated intraperitoneally twice a week.
The mice were monitored daily. On the day of assay termination (see
figure legends) all mice were sacrificed and individual tumor
weights were recorded. No differences were observed between the
various mice groups in their general health at baseline.
Example 1. Generation and Selection of Anti-Nectin-4 mAbs
[0367] The immunogen (Nec4-Fc) expression technology is based on
mammalian HEK 293T cells, a method of choice especially in the case
of glycoproteins that gives the best quality, stability, solubility
and yield. Nec4-Fc protein which is a fusion protein of the
ectodomain of Nectin-4 and human IgG1 Fc domain was produces
recombinantly and purified as following:
[0368] The coding sequence of human Nectin4 was cloned as a fusion
to the Fc fragment of human IgG1 to generate the recombinant
Fc-fusion protein. The ectodomain (extracellular) part of the human
Nectin4 molecule, spanning residues 32 to 349 was used. C-terminal
Serine residue at position 349 of the Nectin4 amino acid sequence
was fused to the heavy chain hinge of a deglycosyltaed Fc (N297A)
of human IgG1, followed by the CH2 and CH3 constant regions. The
open reading frame (ORF) of the recombinant protein was codon
optimized for high-level expression in mammalian cells. The
optimized DNA sequence was produced by GeneArt synthesis service
(Invitrogen) with the addition of flanking DNA sequences
corresponding to EcoRI and NotI restriction sites at the 5' and 3'
ends of the DNA fragment, respectively. The expression vector was
constructed by double digestion of the optimized DNA fragment with
EcoRI and NotI, followed by its ligation into pIRESpuro3 (Clontech
Laboratories, Inc.). The resulting constructs were transfected into
HEK-293T cells by using the FuGENE 6 Transfection Reagent (Roche
Diagnostics). After 48 h, transfected cells were subjected to
antibiotic selection with 5 g/mL puromycin (Sigma-Aldrich). Stable
pools were analyzed for protein secretion by SDS/PAGE. Supernatants
were collected and purified on a Poros 20 protein G column in the
High-Pressure Perfusion Chromatography Station, BioCAD (PerSeptive
Biosystems). The resulted fusion protein immunogen is denoted
Nectin4-Fc.
[0369] For immunization, BALB/c mice were injected with 50 .mu.g of
the immunogen in complete Freund's adjuvant (CFA) followed by 50
.mu.g of the immunogen in incomplete Freund's adjuvant (IFA) at day
14 post first immunization. Next, the sera were analyzed for
anti-Nec-4-Fc antibody titer by ELISA. The mice with the highest
titer were boosted with the 50 .mu.g of the immunogen in PBS. After
three days, the spleen of immune mice was taken and, after lysis of
red blood cells, the splenocytes were fused with SP2/0 cell line.
The potential hybridoma cells were seeded in 20% RPMI 1640 medium
containing hypoxanthine, aminopterin, and thymidine (HAT) for
selection of stable hybridoma cell lines. The abovementioned
procedure was repeated twice and in total, 1034 wells were screened
for anti-Nec4-Fc antibody secretion by ELISA. Next, 30 wells that
their supernatant where positive for binding to Nec4-Fc coated on
ELISA plates, were retested for their positivity, and in parallel a
cross-reactivity test was performed on an irrelevant Fc fusion
protein. This resulted in 10 hybridoma cell lines that secreted
antibodies specifically recognizing Nectin-4 ectodomain. All these
candidates that showed the specific signal in ELISA were tested for
their ability to recognize a native human Nectin-4 protein on
transfectant cell lines.
[0370] RAJI Burkitt's lymphoma cells were transfected with human
Nectin4 and analyzed by FACS for determining the binding of the
five hybridoma clones indicated. The cells were incubated with
different clones and then incubated with human TIGIT-Ig and stained
with a secondary antibody. The Mean Fluorescence Intensity (MFI)
values of the FACS staining are depicted in FIG. 2. As shown,
monoclonal antibodies produced by clones hNec4.01 (Clone 1) and
hNec4.05 (Clone 5), exhibited the best blocking capability of
TIGIT-Nectin4 interactions.
[0371] Similar assays, using JEG3 and LNCap cell lines that
naturally expressed Nectin4 were also performed and have yielded
similar results.
[0372] Five antibodies showed positive staining Out of those 5, 4
were further selected for their IgG isotype (and not IgM isotype)
Finally, the 4 remaining candidates all showed a strong binding
capacity to the native human Nectin-4 molecules expressed on the
surface of live cells and were repetitively tested on several
irrelevant fusion proteins to select for those with zero
cross-reactivity between Nectin-4 and other ligands of the immune
cell receptors. However, only 2 of those were shown to be blocking
antibodies against Nectin-4. Thus, 2 stable clonal cell lines,
hNec4.01 and hNec4.05, have been generated, both of the isotype
kappa IgG1. Next, a large-scale Ab production was performed and
both monoclonal antibodies were purified from the serum free
medium, using GE AKTA Prime Plus Liquid Chromatography System and
HiTrap Protein G columns, in an amount of few milligrams.
[0373] Similarly to the above, additional clones were identified,
out of which clone 11 (hNec4.11) had the best binding and blocking
capacities and thus was screened in parallel to hNec4.05 as
described below.
[0374] Based on the above attempts, it was concluded that even in
the case of an immunogen closely resembling the target protein
Nectin-4 (in terms that it is a dimer, glycosylated by mammalian
cell machinery and produced under non-denaturing conditions), the
chance of obtaining a hybridoma cell line secreting blocking
anti-Nectin 4 antibodies is less than 2% (per mille).
Example 2. Affinity of the Anti-Nectin-4 mAbs to Human Nectin4
[0375] The affinity of antibodies hNec4.01 and hNec4.05 to
fluorophore-labeled human Nectin4-Ig molecule was further
determined using microscale thermophoresis assay (Wienken et al.
2010, Nat. Commun.1:100). Measurements were repeated with at least
three independent protein preparations. As shown in FIG. 3, very
high binding affinities were observed for both antibodies. For
clone hNec4.05 the calculated Kd is 272.+-.154 pM, and for the
hNec4.01 clone the calculated Kd was 107.+-.115 pM.
Example 3. Sequencing of the Anti-Nectin-4 mAbs
[0376] The two hybridoma clones, No. 0.1 and No. 0.5, which
demonstrated the best inhibition of Nectin4-TIGIT binding, were
sent for nucleotide and amino acid sequencing.
Methods
[0377] Total RNA was isolated from the hybridoma cells following
the technical manual of TRIzol.RTM. Reagent (Ambion, Cat. No.:
15596-026). Total RNA was then reverse transcribed into cDNA using
isotype-specific anti-sense primers or universal primers following
the technical manual of PrimeScript.TM. 1 st Strand cDNA Synthesis
Kit (Takara, Cat. No.: 6110A). The antibody fragments of V.sub.H
and V.sub.L were amplified according to the standard operating
procedure (SOP) of rapid amplification of cDNA ends (RACE) of
GenScript. Amplified antibody fragments were cloned into a standard
cloning vector separately. Colony PCR was performed to screen for
clones with inserts of correct sizes. No less than five colonies
with inserts of correct sizes were sequenced for each fragment. The
sequences of different clones were aligned and the consensus
sequence of these clones was provided.
[0378] The following tools for sequence analysis of immunoglobulin
variable regions were used: [0379] i. NCBI Nucleotide BLAST; [0380]
ii. IMGT/V Quest program; and [0381] iii. NCBI IgBLAST. The
obtained sequences are:
TABLE-US-00001 [0381] Clone hNec4.01: Heavy chain variable region
DNA sequence, 414 base pairs (SEQ ID NO: 1): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGCTTCCACTCTGAGGT
CCAACTTCAGCAGTCAGGACCTGAACTGGTGAAACCTGGGGCCTCAGTGAAGATTGCCTGCA
GGGCCTCTGGATACACATTCACTGCCTACAATATCCACTGGGTGAGCCAGAGACATGGAAAG
AGCCTTGAATGGATTGGATATATCTATCCTAACAATGGTGGTTCTGGCTACAACCAGAAATT
CATGAACAAGGCCACATTGACTGTAGACCATTCCTCCAATACAGCCTACATGGAGCTCCGCA
GCCTGACGTCTGAGGACTCTGCAGTCTATTACTGTGCAATATTTGATTACGACGAGGCCTGG
TTTATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Heavy chain variable
region polypeptide sequence, 138 amino acids, (SEQ ID NO: 2):
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MGWSWIFLFLLSGTAGFHSEVQLQQSGPELVKPGASVKIACRASGYTFTAYNIHWVSQRHGK
SLEWIGYIYPNNGGSGYNQKFMNKATLTVDHSSNTAYMELRSLTSEDSAVYYCAIFDYDEAW
FIYWGQGTLVTVSA Heavy chain variable region polypeptide sequence
(FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEQ ID NO: 35)
EVQLQQSGPELVKPGASVKIACRASGYTFTAYNIHWVSQRHGKSLEWIGYIYPNNGGSGYNQ
KFMNKATLTVDHSSNTAYMELRSLTSEDSAVYYCAIFDYDEAWFIYWGQGTLVTVSA Light
chain variable region DNA sequence, 384 base pairs (SEQ ID NO: 3):
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAG
AGGAGAAAATGTTCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCCGGGGAAGAGGTCA
CCATGACCTGTAGTGCCAGCTCAAGTGTTAGTTACATGCACTGGTTCCAGCAGAAGTCAACT
ATCTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCCGGTCGCTT
CAGTGGCAGTGGGTCTGGCAAGTCTTACTCTCTCACGATCAGAAACATGGAGGCTGAAGATG
TTGCCACCTATTACTGTTTTCAGGGGAGTGGGAGCCCGTACACGTTCGGAGGGGGGACCAAG
CTGGAAATTAAA Light chain variable region polypeptide sequence, 128
amino acids (SEQ ID NO: 4): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MDFQVQIFSFLLISASVIMSRGENVLTQSPEIMSASPGEEVTMTCSASSSVSYMHWFQQKST
ISPKLWIYDTSKLASGVPGRFSGSGSGKSYSLTIRNMEAEDVATYYCFQGSGSPYTFGGGTK LEIK
Light chain variable region polypeptide sequence
(FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEQ ID NO: 36)
ENVLTQSPEIMSASPGEEVTMTCSASSSVSYMHWFQQKSTISPKLWIYDTSKLASGVPGRFS
GSGSGKSYSLTIRNMEAEDVATYYCFQGSGSPYTFGGGTKLEIK Clone hNec4.05 Heavy
chain variable region DNA sequence, 408 base pairs (SEQ ID NO: 5):
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGCCAGGT
CCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCA
AGGCCTCTGGCTACACCTTCACAACCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAG
GGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGAACAATGAGAAGTT
CAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCA
GCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAACCCCTATGTTATGGAC
TACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA Heavy chain variable region
polypeptide sequence, 136 amino acids (SEQ ID NO: 6): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTTYYIHWVKQRPGQ
GLEWIGWIYPGNVNTKNNEKFKVKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSNPYVMD
YWGQGTSVTVSS Heavy chain variable region polypeptide sequence
(FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEQ ID NO: 37)
QVQLQQSGPELVKPGASVRISCKASGYTFTTYYIHWVKQRPGQGLEWIGWIYPGNVNTKNNE
KFKVKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSS Light chain
variable region DNA sequence, 381 base pairs (SEQ ID NO: 7): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGTGTCTGGTGCTCATGGGAG
TATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAA
CCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAG
TCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCAC
TGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCGCTGTGCAGGCTGAAGACCTGG
CAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACGTTCGGAGGGGGGACCAAGCTG
GAAATAAAA Light chain variable region polypeptide sequence, 127
amino acids (SEQ ID NO: 8): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKSQTQVFVFLLLCVSGAHGSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQ
SPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISAVQAEDLAVYFCQQDYSSPYTFGGGTKL EIK
Light chain variable region polypeptide sequence
(FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEQ ID NO: 38)
SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRF
TGSGYGTDFTFTISAVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK Clone hNec4.11 Heavy
chain variable region DNA sequence, 408 base pairs (SEQ ID NO: 21):
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGC
CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGAGACTTCAGTG
AAGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGTTACTATATACACTGG
GTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGCTGGATTTATCCTGG
AAATGTTAATACTAAGTATAATGAGAGGTTTAAGGGCAAGGCCACTCTGACTG
CAGACAAATCCTCCAACACAGCCCACATGCAGCTCACCAGCCTGACCTCTGAGG
ACTCTGCGGTCTATTTCTGTGCAAGATCGAACCCCTATGTTATGGACTACTGGG
GTCAAGGAACCTCAGTCACCGTCTCCTCA Heavy chain variable region
polypeptide sequence, 136 amino acids (SEQ ID NO: 22): Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPETSVKISCKASGYTFTSYYIHWVKQR
PGQGLEWIGWIYPGNVNTKYNERFKGKATLTADKSSNTAHMQLTSLTSEDSAVYF
CARSNPYVMDYWGQGTSVTVSS Heavy chain variable region polypeptide
sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEQ ID NO: 39)
QVQLQQSGPELVKPETSVKISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVN
TKYNERFKGKATLTADKSSNTAHMQLTSLTSEDSAVYFCARSNPYVMDYWGQGTS VTVSS Light
chain variable region DNA sequence, 381 base pairs (SEQ ID NO: 23):
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGTGTCTGGTGCTCATGG
GAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGA
GTCACCATAACCTGCAAGGCCAGTCAGAGTGTGAATAATGATGTGGCTTGGTA
TCAACAGAAGCCAGGGCTGTCTCCTGAACTGCTTATGTATTATGCATCCAATCG
CTTCACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACT
TTCACCATCAGCTCTGTGCAGGCTGAAGACCTGGCAATTTATTTCTGTCAGCAGG
CTTATAGGTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATTCAA Light chain
variable region polypeptide sequence, 127 amino acids (SEQ ID NO:
24): Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKSQTQVFVFLLLCVSGAHGSIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQ
QKPGLSPELLMYYASNRFTGVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRS
PYTFGGGTKLEIQ Light chain variable region polypeptide sequence
(FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 (SEO ID NO: 40)
SIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQQKPGLSPELLMYYASNRFT
GVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRSPYTFGGGTKLEIQ
Example 4. Blocking of Nectin4-TIGIT Interactions with Anti-Nectin4
mAb Enhanced NK Cell Killing of Human Cell Lines
[0382] Antibody clones hNec4.05 and hNec4.01 were tested for
blocking Nectin4 binding to TIGIT and inhibiting NK cytotoxicity.
[.sup.35S] methionine-labelled RAJI Burkitt's lymphoma cells
transfected with Nectin4, and LNCap prostate carcinoma cells
(naturally expressing Nectin4), were incubated with 1 .mu.g/well of
either mouse IgG1 as control antibody or mouse anti human Nectin4
mAbs hNec4.01 or hNec4.05. After 1 h the cells were supplemented
with NK cells and incubated for 5 hours. The average specific
killing (.+-.s.d.) in various Effector:Target (E:T) ratios of the
NK:Cancer cells is plotted in FIG. 4A (RAJI cells) and FIG. 4B
(LNCap cells). * indicates significant effect (p<0.05) of the
hNec4.01 and hNec4.05 clones in comparison with the control
antibody. Each figure shows one representative experiment out of
three performed. The same effect was determined when using
MCF-7-breast cancer cell line.
Example 5. Verifying the Specificity of Binding of the Anti-Nectin4
Antibodies
[0383] As tested using FACS staining and demonstrated in FIGS.
5A-5C, the mAbs hNec4.01 and hNec4.05 are specific to human Nectin4
and do not bind the murine protein. RAJI cells were transfected
with murine Nectin4 (black line histograms). First, RAJI cells
transfected with murine Nectin4 lysates was used in Western blot
assay with a commercial anti murine Nectin4 mAb (Clone 356704) in
order to verify murine Nectin4 expression (A). Next, the cells were
stained with 0.2 .mu.g of (B) clone hNec4.01 or (C) clone hNec4.05.
Both hNec4.01 and hNec4.05 mAbs do not show binding to murine
Nectin4.
Example 6. Anti-Nectin4 Antibodies are Able to Block
Nectin4-Nectin1 Interactions
[0384] The ability to the anti-Nectin4 to block Nectin4-Nectin1
interactions was also determined. FACS staining of RAJI cells
transfected with Nectin4 is demonstrated in FIGS. 6A-6C. Cells were
Pre-incubated with 1 .mu.g of (FIG. 6A) clone hNec4.01 or (FIG. 6B)
clone hNec4.05 and then incubated with 3 .mu.g of Nectin1-Ig (black
line). Staining without blocking appears as gray line. Grey filled
histograms are background control staining of secondary antibody
only. It is concluded that the mAbs are able to block
Nectin4-Nectin1 interaction that is suspected to increase
invasiveness of tumors expressing Nectin4.
Example 7. In Vivo Models
[0385] The efficacy of the anti-Nectin4 mAbs was determined in vivo
in animal models. Cell lines that naturally (MDA-MB-453) or
recombinantly (Raji Nectin4 OE) express Nectin4 were injected SC to
mice (5.times.10.sup.6 cells per mouse). SCID beige mice, which
lack NK, B and T cells were used. In order to study NK cell
contribution to tumor cell growth in these models human NK cells at
1.times.10.sup.6 were co injected with the tumor cells in some of
the treatment groups.
[0386] The anti-Nectin4 mAbs clone hNec4.05, or a control Ab (anti
murine CD3, InVivoMAb-clone 17A2) were tested for their in vivo
effect on tumor growth directly, or together with NK cells. The
mAbs were injected IP at 75 .mu.g per mouse, twice a week. Tumor
weights were measured at study termination. As seen in FIG. 7 (A),
overexpression (OE) of Nectin-4 on Raji cells did not affect their
growth in comparison to the growth of their parental cells which
were transfected with empty vector (EV). Nevertheless, in the
presence of human NK cells, OE of nectin4 increased tumor growth
suggesting a negative effect of Nectin4 on NK mediated tumor
suppression activity of NK cells. As seen in FIG. 7B, the addition
of anti-Nectin4 Ab blocked NK cell suppression leading to reduced
tumor growth in comparison to control Ab treated animals Last, and
as can be seen in FIG. 7C these effects were also seen when a cell
line (MDA-MB-453) that naturally express Nectin4 was used. In the
absence of NK cells the Abs had no effect, while the Abs
significantly enhanced the anti-tumor effect of human NK cells co
injected with the tumor cells. Together, these experiments indicate
that anti-Nectin4 antibodies, such as hNec4.05 mAbs, can augment NK
cytotoxicity in vivo and lead to tumor growth inhibition.
Example 8. Binding of Anti-Nectin-4 Clones to Human, Monkey and
Murine Nectin-4 Expressed on Cells
[0387] Binding of murine anti-human Nectin-4 clones hNec4.05 and
hNec4.11 to Nectin-4 expressed on the human cell line MDA-MD-453
was evaluated by FACS analysis. FIG. 8A depicts the EC50 values
that were calculated following titration of Ab binding (range of
20-0.01 nM), and the maximal binding signal for each clone Similar
values were reached when the chimeric versions of the murine Abs
were tested, in which the murine IgG1 Fc chain was replaced with
the human IgG1 Fc one. FIG. 8B depicts antibody binding to CHO
cells transfected with Cynomolgus (Cyno)-Nectin-4 and FIG. 8C
depicts antibody binding to CHO cells transfected with
murine-Nectin-4. These data demonstrate EC50 values to the human
target at the sub-nanomolar range, which are at the scale of the Kd
values presented in FIG. 3 for clones hNec4.01 and hNec4.05.
Additionally, these results demonstrate cross reactivity of these
two clones to the monkey (cynomolgus) Nectin-4 target, as they bind
it with similar EC50 values to the ones calculated for the human
target. This may be important for pre-clinical studies of the Ab.
Last, clone hNec4.11, but not clone hNec4.05, was shown to also
cross react with the murine Nectin-4 target. Yet, in this case the
calculated EC50 was about 10-fold higher than the one calculated
for the human target.
Example 9. Blocking of Nectin-4 Ligands by Anti-Nectin-4 Clones
[0388] Antibody clones hNec4.05 and hNec4.11 block the binding of
Nectin-4 to its ligands TIGIT and Nectin-1. Binding of Nectin-4
ligands was assessed by FACS analysis. CHO cells transfected with
human-Nectin-4 were incubated with either human TIGIT-Ig (FIGS. 9 A
& C) or with human Nectin-1-Ig (FIGS. 9 B & D), both at 20
ug/ml, with or without anti-Nectin-4 clone hNec4.05 (9A & 9B)
or clone hNec4.11 (9C & 9D), both at 8 ug/ml. Robust binding
inhibition by the anti-Nectin-4 Abs is seen in all cases. Remaining
signal is likely due to ligand binding to other receptors expressed
by CHO cells, such as PVR, which are not affected by the
anti-Nectin-4 Abs. These results suggest that Nectin-4 clones
hNec4.05 and hNec4.11 may affect the target cancer cells by
blocking signaling through cell surface Nectin-4. Additionally,
these Abs may also affect effector cells which express Nectin-4
ligands, such as the inhibitory ligand TIGIT.
Example 10. Human IgG1 Chimeric Ab Clones hNec4.05 and hNec4.1
Enhance NK Cell Activation in the Presence of Tumor Cells
[0389] Depicted in FIG. 11 are relative expression levels of the
degranulation marker CD107a on NK cells. Human NK cells (effector,
E) were incubated with the target cells (T) HT1376 (FIG. 10A) and
MDA-MD-453 (FIG. 10B) at E:T ratio of 2:1. Incubation was done in
the presence of 12 ug/ml chimeric clones hNec4.05hIgG1,
hNec4.11hIgG1 or a control hIgG1. After two hours NK cells were
assayed for their degranulation and activation status by FACS
analysis of CD107a expression. Degranulation of NK cells in
presence of control hIgG1 was set as 1 and fold induction was
calculated accordingly. Shown are averages of 2-3 repeats and their
normalized SDs. These results suggest an anti-cancer effect for
these Abs by enhancing anti-tumor NK cell activity. Slight
advantage of clone hNec4.11-hIgG1 over clone hNec4.05hIgG1 in ADCC
induction was shown, which is in line with the higher max binding
of clone hNec4.11 as shown in FIG. 8A.
Example 11. CAR-T Driven by hNec4.11 Lead to Specific T Cell
Activation in the Presence of Tumor Cells Expressing Nectin-4
[0390] A schematic drawing of the CAR-T construct is seen in FIG.
11A. The transduction efficacy as judged by GFP expression was
above 99% (FIG. 11B). Parental Jurkat cells or Jurkat cells
expressing the CAR-T construct with hNec4.11-based single chain
variable region (scFV) (Jurkat pHAGE2.4.11) were incubated with the
target cells HT1376 and MDA-MD-453 (MDA-453) (FIG. 11C). The
secretion of IL-2 by the Jurkat cells was significantly induced by
the CAR-T expression. Next, PBMCs were transduced using pHAGE2.4.11
lenti particles (FIG. 11D). CAR-T PBMCs were incubated with HT1376
cells through a range of E:Ts. After 48 hours the effector cells
were removed, and target cell viability was assessed using
CellTiter-Glo.RTM. Luminescent Cell Viability Assay. The killing of
the target cells was significant. Taken together these observations
point to the possibility of further developing CAR-T therapy that
is based on the nectin4 Abs described herein.
TABLE-US-00002 ScFv sequences of CAR construct Clone 11-Nucleic
acids (SEQ ID NO: 31)
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTG
TGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCC
TGAGACTTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACA
AGTTACTATATACACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAGT
GGATTGGCTGGATTTATCCTGGAAATGTTAATACTAAGTATAATGAGAG
GTTTAAGGGCAAGGCCACTCTGACTGCAGACAAATCCTCCAACACAGCC
CACATGCAGCTCACCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCT
GTGCAAGATCGAACCCCTATGTTATGGACTACTGGGGTCAAGGAACCTC
AGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGAGGAGGTGGTTCTGGA
GGAGGTGGTTCTGATATCGTGATGACCCAGACTCCCAAATTCCTGCTTG
TATCAGCAGGAGACAGAGTCACCATAACCTGCAAGGCCAGTCAGAGTGT
GAATAATGATGTGGCTTGGTATCAACAGAAGCCAGGGCTGTCTCCTGAA
CTGCTTATGTATTATGCATCCAATCGCTTCACTGGAGTCCCTGATCGCT
TCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCTCTGT
GCAGGCTGAAGACCTGGCAATTTATTTCTGTCAGCAGGCTTATAGGTCT
CCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATTCAA Clone 11-Amino acids (SEQ
ID NO: 32) MGWSCIILFLVATATGVHSQVQLQQSGPELVKPETSVKISCKASGYTFT
SYYIHWVKQRPGQGLEWIGWIYPGNVNTKYNERFKGKATLTADKSSNTA
HMQLTSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSSGGGGSGGGGSG
GGGSDIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQQKPGLSPE
LLMYYASNRFTGVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRS PYTFGGGTKLEIQ
Clone 5-Nucleic acids (SEQ ID NO: 33)
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTG
TGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCC
TGGGGCTTCAGTGAGGATATCCTGCAAGGCCTCTGGCTACACCTTCACA
ACCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGT
GGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGAACAATGAGAA
GTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCC
TACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCT
GTGCAAGATCGAACCCCTATGTTATGGACTACTGGGGTCAGGGAACCTC
AGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGAGGAGGTGGTTCTGGA
GGAGGTGGTTCTAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTG
TATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGT
GAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAA
CTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCT
TCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCGCTGT
GCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCT
CCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Clone 5-Amino acids (SEQ ID
NO: 34) MGWSCIILFLVATATGVHSQVQLQQSGPELVKPGASVRISCKASGYTFT
TYYIHWVKQRPGQGLEWIGWIYPGNVNTKNNEKFKVKATLTADKSSSTA
YMQLSSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSSGGGGSGGGGSG
GGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPK
LLIYYASNRYTGVPDRFTGSGYGTDFTFTISAVQAEDLAVYFCQQDYSS PYTFGGGTKLEIK
[0391] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation.
Sequence CWU 1
1
461414DNAMus musculus 1atgggatgga gctggatctt tctcttcctc ctgtcaggaa
ctgcaggctt ccactctgag 60gtccaacttc agcagtcagg acctgaactg gtgaaacctg
gggcctcagt gaagattgcc 120tgcagggcct ctggatacac attcactgcc
tacaatatcc actgggtgag ccagagacat 180ggaaagagcc ttgaatggat
tggatatatc tatcctaaca atggtggttc tggctacaac 240cagaaattca
tgaacaaggc cacattgact gtagaccatt cctccaatac agcctacatg
300gagctccgca gcctgacgtc tgaggactct gcagtctatt actgtgcaat
atttgattac 360gacgaggcct ggtttattta ctggggccaa gggactctgg
tcactgtctc tgca 4142138PRTMus musculus 2Met Gly Trp Ser Trp Ile Phe
Leu Phe Leu Leu Ser Gly Thr Ala Gly1 5 10 15Phe His Ser Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val
Lys Ile Ala Cys Arg Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ala Tyr Asn
Ile His Trp Val Ser Gln Arg His Gly Lys Ser Leu 50 55 60Glu Trp Ile
Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn65 70 75 80Gln
Lys Phe Met Asn Lys Ala Thr Leu Thr Val Asp His Ser Ser Asn 85 90
95Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110Tyr Tyr Cys Ala Ile Phe Asp Tyr Asp Glu Ala Trp Phe Ile
Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ala 130
1353384DNAMus musculus 3atggattttc aagtgcagat tttcagcttc ctgctaatca
gtgcctcagt cataatgtcc 60agaggagaaa atgttctcac ccagtctcca gaaatcatgt
ctgcatctcc cggggaagag 120gtcaccatga cctgtagtgc cagctcaagt
gttagttaca tgcactggtt ccagcagaag 180tcaactatct cccccaaact
ctggatttat gacacatcca aactggcttc tggagtcccc 240ggtcgcttca
gtggcagtgg gtctggcaag tcttactctc tcacgatcag aaacatggag
300gctgaagatg ttgccaccta ttactgtttt caggggagtg ggagcccgta
cacgttcgga 360ggggggacca agctggaaat taaa 3844128PRTMus musculus
4Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5
10 15Val Ile Met Ser Arg Gly Glu Asn Val Leu Thr Gln Ser Pro Glu
Ile 20 25 30Met Ser Ala Ser Pro Gly Glu Glu Val Thr Met Thr Cys Ser
Ala Ser 35 40 45Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Ser
Thr Ile Ser 50 55 60Pro Lys Leu Trp Ile Tyr Asp Thr Ser Lys Leu Ala
Ser Gly Val Pro65 70 75 80Gly Arg Phe Ser Gly Ser Gly Ser Gly Lys
Ser Tyr Ser Leu Thr Ile 85 90 95Arg Asn Met Glu Ala Glu Asp Val Ala
Thr Tyr Tyr Cys Phe Gln Gly 100 105 110Ser Gly Ser Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 1255408DNAMus musculus
5atgggatgga gccggatctt tctcttcctc ctgtcaataa ttgcaggtgt ccattgccag
60gtccagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaggatatcc
120tgcaaggcct ctggctacac cttcacaacc tactatatac actgggtgaa
gcagaggcct 180ggacagggac ttgagtggat tggatggatt tatcctggaa
atgttaatac taagaacaat 240gagaagttca aggtcaaggc cacactgact
gcagacaaat cctccagcac agcctacatg 300cagctcagca gcctgacctc
tgaggactct gcggtctatt tctgtgcaag atcgaacccc 360tatgttatgg
actactgggg tcagggaacc tcagtcaccg tctcctca 4086136PRTMus musculus
6Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Ile Ala Gly1 5
10 15Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
Lys 20 25 30Pro Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Thr Thr Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn
Thr Lys Asn Asn65 70 75 80Glu Lys Phe Lys Val Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Phe Cys Ala Arg Ser Asn
Pro Tyr Val Met Asp Tyr Trp Gly Gln 115 120 125Gly Thr Ser Val Thr
Val Ser Ser 130 1357381DNAMus musculus 7atgaagtcac agacccaggt
cttcgtattt ctactgctct gtgtgtctgg tgctcatggg 60agtattgtga tgacccagac
tcccaaattc ctgcttgtat cagcaggaga cagggttacc 120ataacctgca
aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca
180gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg
agtccctgat 240cgcttcactg gcagtggata tgggacggat ttcactttca
ccatcagcgc tgtgcaggct 300gaagacctgg cagtttattt ctgtcagcag
gattatagct ctccgtacac gttcggaggg 360gggaccaagc tggaaataaa a
3818127PRTMus musculus 8Met Lys Ser Gln Thr Gln Val Phe Val Phe Leu
Leu Leu Cys Val Ser1 5 10 15Gly Ala His Gly Ser Ile Val Met Thr Gln
Thr Pro Lys Phe Leu Leu 20 25 30Val Ser Ala Gly Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Ser 35 40 45Val Ser Asn Asp Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60Lys Leu Leu Ile Tyr Tyr Ala
Ser Asn Arg Tyr Thr Gly Val Pro Asp65 70 75 80Arg Phe Thr Gly Ser
Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95Ala Val Gln Ala
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr 100 105 110Ser Ser
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120
12595PRTMus musculus 9Ala Tyr Asn Ile His1 51017PRTMus musculus
10Tyr Ile Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn Gln Lys Phe Met1
5 10 15Asn1110PRTMus musculus 11Phe Asp Tyr Asp Glu Ala Trp Phe Ile
Tyr1 5 101210PRTMus musculus 12Ser Ala Ser Ser Ser Val Ser Tyr Met
His1 5 10137PRTMus musculus 13Asp Thr Ser Lys Leu Ala Ser1
5149PRTMus musculus 14Phe Gln Gly Ser Gly Ser Pro Tyr Thr1
5155PRTMus musculus 15Thr Tyr Tyr Ile His1 51617PRTMus musculus
16Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Asn Asn Glu Lys Phe Lys1
5 10 15Val178PRTMus musculus 17Ser Asn Pro Tyr Val Met Asp Tyr1
51811PRTMus musculus 18Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala1
5 10197PRTMus musculus 19Tyr Ala Ser Asn Arg Tyr Thr1 5209PRTMus
musculus 20Gln Gln Asp Tyr Ser Ser Pro Tyr Thr1 521408DNAMus
musculus 21atgggatgga gccggatctt tctcttcctc ctgtcaataa ttgcaggtgt
ccattgccag 60gtccagctgc agcagtctgg acctgaactg gtgaagcctg agacttcagt
gaagatatcc 120tgcaaggctt ctggctacac cttcacaagt tactatatac
actgggtgaa acagaggcct 180ggacagggac ttgagtggat tggctggatt
tatcctggaa atgttaatac taagtataat 240gagaggttta agggcaaggc
cactctgact gcagacaaat cctccaacac agcccacatg 300cagctcacca
gcctgacctc tgaggactct gcggtctatt tctgtgcaag atcgaacccc
360tatgttatgg actactgggg tcaaggaacc tcagtcaccg tctcctca
40822136PRTMus musculus 22Met Gly Trp Ser Arg Ile Phe Leu Phe Leu
Leu Ser Ile Ile Ala Gly1 5 10 15Val His Cys Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Glu Thr Ser Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Tyr Ile His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Trp Ile
Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn65 70 75 80Glu Arg Phe Lys
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn 85 90 95Thr Ala His
Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr
Phe Cys Ala Arg Ser Asn Pro Tyr Val Met Asp Tyr Trp Gly Gln 115 120
125Gly Thr Ser Val Thr Val Ser Ser 130 13523381DNAMus musculus
23atgaagtcac agacccaggt cttcgtattt ctactgctct gtgtgtctgg tgctcatggg
60agtattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagagtcacc
120ataacctgca aggccagtca gagtgtgaat aatgatgtgg cttggtatca
acagaagcca 180gggctgtctc ctgaactgct tatgtattat gcatccaatc
gcttcactgg agtccctgat 240cgcttcactg gcagtggata tgggacggat
ttcactttca ccatcagctc tgtgcaggct 300gaagacctgg caatttattt
ctgtcagcag gcttataggt ctccgtacac gttcggaggg 360gggaccaagc
tggaaattca a 38124127PRTMus musculus 24Met Lys Ser Gln Thr Gln Val
Phe Val Phe Leu Leu Leu Cys Val Ser1 5 10 15Gly Ala His Gly Ser Ile
Val Met Thr Gln Thr Pro Lys Phe Leu Leu 20 25 30Val Ser Ala Gly Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser 35 40 45Val Asn Asn Asp
Val Ala Trp Tyr Gln Gln Lys Pro Gly Leu Ser Pro 50 55 60Glu Leu Leu
Met Tyr Tyr Ala Ser Asn Arg Phe Thr Gly Val Pro Asp65 70 75 80Arg
Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90
95Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Phe Cys Gln Gln Ala Tyr
100 105 110Arg Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Gln 115 120 125255PRTMus musculus 25Ser Tyr Tyr Ile His1
52617PRTMus musculus 26Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr
Asn Glu Arg Phe Lys1 5 10 15Gly278PRTMus musculus 27Ser Asn Pro Tyr
Val Met Asp Tyr1 52811PRTMus musculus 28Lys Ala Ser Gln Ser Val Asn
Asn Asp Val Ala1 5 10297PRTMus musculus 29Tyr Ala Ser Asn Arg Phe
Thr1 5309PRTMus musculus 30Gln Gln Ala Tyr Arg Ser Pro Tyr Thr1
531774DNAArtificial SequenceSynthetic sequence 31atgggatgga
gctgtatcat cctcttcttg gtagcaacag ctacaggtgt gcattcacag 60gtccagctgc
agcagtctgg acctgaactg gtgaagcctg agacttcagt gaagatttcc
120tgcaaggctt ctggctacac cttcacaagt tactatatac actgggtgaa
acagaggcct 180ggacagggac ttgagtggat tggctggatt tatcctggaa
atgttaatac taagtataat 240gagaggttta agggcaaggc cactctgact
gcagacaaat cctccaacac agcccacatg 300cagctcacca gcctgacctc
tgaggactct gcggtctatt tctgtgcaag atcgaacccc 360tatgttatgg
actactgggg tcaaggaacc tcagtcaccg tctcctcagg tggaggtggc
420tccggaggag gtggttctgg aggaggtggt tctgatatcg tgatgaccca
gactcccaaa 480ttcctgcttg tatcagcagg agacagagtc accataacct
gcaaggccag tcagagtgtg 540aataatgatg tggcttggta tcaacagaag
ccagggctgt ctcctgaact gcttatgtat 600tatgcatcca atcgcttcac
tggagtccct gatcgcttca ctggcagtgg atatgggacg 660gatttcactt
tcaccatcag ctctgtgcag gctgaagacc tggcaattta tttctgtcag
720caggcttata ggtctccgta cacgttcgga ggggggacca agctggaaat tcaa
77432258PRTArtificial SequenceSynthetic sequence 32Met Gly Trp Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Glu
Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr
Ser Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55
60Glu Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn65
70 75 80Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn 85 90 95Thr Ala His Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser
Ala Val 100 105 110Tyr Phe Cys Ala Arg Ser Asn Pro Tyr Val Met Asp
Tyr Trp Gly Gln 115 120 125Gly Thr Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Asp
Ile Val Met Thr Gln Thr Pro Lys145 150 155 160Phe Leu Leu Val Ser
Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala 165 170 175Ser Gln Ser
Val Asn Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly 180 185 190Leu
Ser Pro Glu Leu Leu Met Tyr Tyr Ala Ser Asn Arg Phe Thr Gly 195 200
205Val Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe
210 215 220Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Phe
Cys Gln225 230 235 240Gln Ala Tyr Arg Ser Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu 245 250 255Ile Gln33774DNAArtificial
SequenceSynthetic sequence 33atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctacaggtgt gcattcacag 60gtccagctgc agcagtctgg acctgagctg
gtgaagcctg gggcttcagt gaggatatcc 120tgcaaggcct ctggctacac
cttcacaacc tactatatac actgggtgaa gcagaggcct 180ggacagggac
ttgagtggat tggatggatt tatcctggaa atgttaatac taagaacaat
240gagaagttca aggtcaaggc cacactgact gcagacaaat cctccagcac
agcctacatg 300cagctcagca gcctgacctc tgaggactct gcggtctatt
tctgtgcaag atcgaacccc 360tatgttatgg actactgggg tcagggaacc
tcagtcaccg tctcctcagg tggaggtggc 420tccggaggag gtggttctgg
aggaggtggt tctagtattg tgatgaccca gactcccaaa 480ttcctgcttg
tatcagcagg agacagggtt accataacct gcaaggccag tcagagtgtg
540agtaatgatg tagcttggta ccaacagaag ccagggcagt ctcctaaact
gctgatatac 600tatgcatcca atcgctacac tggagtccct gatcgcttca
ctggcagtgg atatgggacg 660gatttcactt tcaccatcag cgctgtgcag
gctgaagacc tggcagttta tttctgtcag 720caggattata gctctccgta
cacgttcgga ggggggacca agctggaaat aaaa 77434258PRTArtificial
SequenceSynthetic sequence 34Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Gln Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val Arg Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Thr Tyr Tyr Ile His
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Trp
Ile Tyr Pro Gly Asn Val Asn Thr Lys Asn Asn65 70 75 80Glu Lys Phe
Lys Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105
110Tyr Phe Cys Ala Arg Ser Asn Pro Tyr Val Met Asp Tyr Trp Gly Gln
115 120 125Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly 130 135 140Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr
Gln Thr Pro Lys145 150 155 160Phe Leu Leu Val Ser Ala Gly Asp Arg
Val Thr Ile Thr Cys Lys Ala 165 170 175Ser Gln Ser Val Ser Asn Asp
Val Ala Trp Tyr Gln Gln Lys Pro Gly 180 185 190Gln Ser Pro Lys Leu
Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly 195 200 205Val Pro Asp
Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe 210 215 220Thr
Ile Ser Ala Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln225 230
235 240Gln Asp Tyr Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu 245 250 255Ile Lys35119PRTMus musculus 35Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile
Ala Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30Asn Ile His
Trp Val Ser Gln Arg His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr
Ile Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn Gln Lys Phe 50 55 60Met
Asn Lys Ala Thr Leu Thr Val Asp His Ser Ser Asn Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Ile Phe Asp Tyr Asp Glu Ala Trp Phe Ile Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ala 11536106PRTMus musculus
36Glu Asn Val Leu Thr Gln Ser Pro Glu Ile Met Ser Ala Ser Pro Gly1
5 10 15Glu Glu Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30His Trp Phe Gln Gln Lys Ser Thr Ile Ser Pro Lys Leu Trp
Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Lys Ser Tyr Ser Leu Thr Ile Arg Asn
Met Glu Ala Glu65 70 75 80Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly
Ser Gly Ser Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 10537117PRTMus musculus 37Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Arg Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30Tyr Ile His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile
Tyr Pro Gly Asn Val Asn Thr Lys Asn Asn Glu Lys Phe 50 55 60Lys Val
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95Ala Arg Ser Asn Pro Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr
Ser 100 105 110Val Thr Val Ser Ser 11538107PRTMus musculus 38Ser
Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ala
Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp
Tyr Ser Ser Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10539117PRTMus musculus 39Gln Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro Glu Thr1 5 10 15Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Ile His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro
Gly Asn Val Asn Thr Lys Tyr Asn Glu Arg Phe 50 55 60Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala His65 70 75 80Met Gln
Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala
Arg Ser Asn Pro Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105
110Val Thr Val Ser Ser 11540107PRTMus musculus 40Ser Ile Val Met
Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asn Asn Asp 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Leu Ser Pro Glu Leu Leu Met 35 40 45Tyr
Tyr Ala Ser Asn Arg Phe Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55
60Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65
70 75 80Glu Asp Leu Ala Ile Tyr Phe Cys Gln Gln Ala Tyr Arg Ser Pro
Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Gln 100
10541357DNAMus musculus 41gaggtccaac ttcagcagtc aggacctgaa
ctggtgaaac ctggggcctc agtgaagatt 60gcctgcaggg cctctggata cacattcact
gcctacaata tccactgggt gagccagaga 120catggaaaga gccttgaatg
gattggatat atctatccta acaatggtgg ttctggctac 180aaccagaaat
tcatgaacaa ggccacattg actgtagacc attcctccaa tacagcctac
240atggagctcc gcagcctgac gtctgaggac tctgcagtct attactgtgc
aatatttgat 300tacgacgagg cctggtttat ttactggggc caagggactc
tggtcactgt ctctgca 35742318DNAMus musculus 42gaaaatgttc tcacccagtc
tccagaaatc atgtctgcat ctcccgggga agaggtcacc 60atgacctgta gtgccagctc
aagtgttagt tacatgcact ggttccagca gaagtcaact 120atctccccca
aactctggat ttatgacaca tccaaactgg cttctggagt ccccggtcgc
180ttcagtggca gtgggtctgg caagtcttac tctctcacga tcagaaacat
ggaggctgaa 240gatgttgcca cctattactg ttttcagggg agtgggagcc
cgtacacgtt cggagggggg 300accaagctgg aaattaaa 31843357DNAMus
musculus 43gaggtccaac ttcagcagtc aggacctgaa ctggtgaaac ctggggcctc
agtgaagatt 60gcctgcaggg cctctggata cacattcact gcctacaata tccactgggt
gagccagaga 120catggaaaga gccttgaatg gattggatat atctatccta
acaatggtgg ttctggctac 180aaccagaaat tcatgaacaa ggccacattg
actgtagacc attcctccaa tacagcctac 240atggagctcc gcagcctgac
gtctgaggac tctgcagtct attactgtgc aatatttgat 300tacgacgagg
cctggtttat ttactggggc caagggactc tggtcactgt ctctgca 35744321DNAMus
musculus 44agtattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga
cagggttacc 60ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca
acagaagcca 120gggcagtctc ctaaactgct gatatactat gcatccaatc
gctacactgg agtccctgat 180cgcttcactg gcagtggata tgggacggat
ttcactttca ccatcagcgc tgtgcaggct 240gaagacctgg cagtttattt
ctgtcagcag gattatagct ctccgtacac gttcggaggg 300gggaccaagc
tggaaataaa a 32145351DNAMus musculus 45caggtccagc tgcagcagtc
tggacctgaa ctggtgaagc ctgagacttc agtgaagata 60tcctgcaagg cttctggcta
caccttcaca agttactata tacactgggt gaaacagagg 120cctggacagg
gacttgagtg gattggctgg atttatcctg gaaatgttaa tactaagtat
180aatgagaggt ttaagggcaa ggccactctg actgcagaca aatcctccaa
cacagcccac 240atgcagctca ccagcctgac ctctgaggac tctgcggtct
atttctgtgc aagatcgaac 300ccctatgtta tggactactg gggtcaagga
acctcagtca ccgtctcctc a 35146321DNAMus musculus 46agtattgtga
tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagagtcacc 60ataacctgca
aggccagtca gagtgtgaat aatgatgtgg cttggtatca acagaagcca
120gggctgtctc ctgaactgct tatgtattat gcatccaatc gcttcactgg
agtccctgat 180cgcttcactg gcagtggata tgggacggat ttcactttca
ccatcagctc tgtgcaggct 240gaagacctgg caatttattt ctgtcagcag
gcttataggt ctccgtacac gttcggaggg 300gggaccaagc tggaaattca a 321
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